In vivo incorporation of unnatural amino acids

ABSTRACT

The invention provides methods and compositions for in vivo incorporation of unnatural amino acids. Also provided are compositions including proteins with unnatural amino acids.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 11/254,161, filed Oct.18, 2005, which is a continuation of U.S. patent application Ser. No.10/126,927, filed Apr. 19, 2002, now U.S. Pat. No. 7,045,337, and claimspriority to and benefit of U.S. provisional patent application Ser. No.60/285,030, filed Apr. 19, 2001, and U.S. provisional patent applicationSer. No. 60/355,514, filed Feb. 6, 2002, the specifications of which areincorporated herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

The invention was made with United States Government support under GrantNo. N0001498F0402 from the Office of Naval Research, Grant No. GM62159from the National Institutes of Health, and Contract Nos.DE-FG03-00ER45812, DE-AC-3-76SF00098 from the Department of Energy. TheUnited States Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to the field of protein biochemistry. Inparticular, the invention relates to the field of compositions andmethods for producing proteins that include unnatural amino acids.

BACKGROUND OF THE INVENTION

Proteins carry out virtually all of the complex processes of life, fromphotosynthesis to signal transduction and the immune response. Tounderstand and control these intricate activities, a betterunderstanding of the relationship between the structure and function ofproteins is needed.

Unlike small organic molecule synthesis wherein almost any structuralchange can be made to influence functional properties of a compound, thesynthesis of proteins is limited to changes encoded by the twentynatural amino acids. The genetic code of every known organism, frombacteria to human, encodes the same twenty common amino acids. Theseamino acids can be modified by posttranslational modification ofproteins, e.g., glycosylation, phosphorylation or oxidation, or in rarerinstances, by the enzymatic modification of aminoacylated suppressortRNAs, e.g., in the case of selenocysteine. Nonetheless, polypeptides,which are synthesized from only these 20 simple building blocks, carryout all of the complex processes of life.

Both site-directed and random mutagenesis, in which specific amino acidsin a protein can be replaced with any of the other nineteen common aminoacids, have become important tools for understanding the relationshipbetween the structure and function of proteins. These methodologies havemade possible the generation of proteins with enhanced properties,including stability, catalytic activity and binding specificity.Nevertheless, changes in proteins are limited to the 20 common aminoacids, most of which have simple functional groups. See Knowles, J. R.Tinkering with enzymes: what are we learning? Science, 236(4806)1252-1258 (1987); and, Zoller, M. J., Smith, M. Oligonucleotide-directedmutagenesis of DNA fragments cloned into M13 vectors, Methods Enzymol,154:468-500 (1983). By expanding the genetic code to include additionalamino acids with novel biological, chemical or physical properties, theproperties of proteins, e.g., the size, acidity, nucleophilicity,hydrogen-bonding, hydrophobic properties, can be modified as compared toa protein composed of only amino acids from the 20 common amino acids,e.g., as in a naturally occurring protein.

Several strategies have been employed to introduce unnatural amino acidsinto proteins. The first experiments involved the derivatization ofamino acids with reactive side-chains such as Lys, Cys and Tyr, forexample, the conversion of lysine to N²-acetyl-lysine. Chemicalsynthesis also provides a straightforward method to incorporateunnatural amino acids, but routine solid-phase peptide synthesis isgenerally limited to small peptides or proteins. With less than 100residues. With the recent development of enzymatic ligation and nativechemical ligation of peptide fragments, it is possible to make largerproteins, but the method is not easily scaled. See, e.g., P. E. Dawsonand S. B. H. Kent, Annu. Rev. Biochem., 69:923 (2000). A general invitro biosynthetic method in which a suppressor tRNA chemically acylatedwith the desired unnatural amino acid is added to an in vitro extractcapable of supporting protein biosynthesis, has been used tosite-specifically incorporate over 100 unnatural amino acids into avariety of proteins of virtually any size. See, e.g., V. W. Cornish, D.Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl., 1995, 34:621(1995); C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins, Science 244 182-188 (1989); and, J. D. Bain,C. G. Glabe, T. A. Dix, A. R. Chamberlin, E. S. Diala, Biosyntheticsite-specific incorporation of a non-natural amino acid into apolypeptide, J. Am. Chem. Soc. 111 8013-8014 (1989). A broad range offunctional groups has been introduced into proteins for studies ofprotein stability, protein folding, enzyme mechanism, and signaltransduction. Although these studies demonstrate that the proteinbiosynthetic machinery tolerates a wide variety of amino acid sidechains, the method is technically demanding, and yields of mutantproteins are low.

Over 50 years ago, it was found that many analogs of natural amino acidsinhibit the growth of bacteria. Analysis of the proteins produced in thepresence of these amino acid analogs revealed that they had beensubstituted for their natural counterparts, to various extents. See,e.g., M. H. Richmond, Bacteriol. Rev., 26:398 (1962). This occursbecause the aminoacyl-tRNA synthetase, the enzyme responsible for theattachment of the correct amino acid to its cognate tRNA, cannotrigorously distinguish the analog from the corresponding natural aminoacid. For instance, norleucine is charged by methionyl-tRNA synthetase,and p-fluorophenylalanine is charged by phenylalanine-tRNA synthetase.See, D. B. Cowie, G. N. Cohen, E. T. Bolton and H.DeRrobinchon-Szulmajst, Biochim. Biophys. Acta, 1959, 34:39 (1959); and,R. Munier and G. N. Cohen, Biochim. Biophys. Acta, 1959, 31:378 (1959).

An in vivo method, termed selective pressure incorporation, was laterdeveloped to exploit the promiscuity of wild-type synthetases. See,e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dong, L.Moroder and R. Huber, FASEB J., 13:41 (1999). An auxotrophic strain, inwhich the relevant metabolic pathway supplying the cell with aparticular natural amino acid is switched off, is grown in minimal mediacontaining limited concentrations of the natural amino acid, whiletranscription of the target gene is repressed. At the onset of astationary growth phase, the natural amino acid is depleted and replacedwith the unnatural amino acid analog. Induction of expression of therecombinant protein results in the accumulation of a protein containingthe unnatural analog. For example, using this strategy, o, m andp-fluorophenylalanines have been incorporated into proteins, and exhibittwo characteristic shoulders in the UV spectrum which can be easilyidentified, see, e.g., C. Minks, R. Huber, L. Moroder and N. Budisa,Anal. Biochem., 284:29 (2000); trifluoromethionine has been used toreplace methionine in bacteriophage T4 lysozyme to study its interactionwith chitooligosaccharide ligands by ¹⁹F NMR, see, e.g., H. Duewel, E.Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); andtrifluoroleucine has been inserted in place of leucine, resulting inincreased thermal and chemical stability of a leucine-zipper protein.See, e.g., Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F.DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40:1494 (2001).Moreover, selenomethionine and telluromethionine are incorporated intovarious recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, e.g., W. A. Hendrickson, J. R. Horton and D.M. Lemaster, EMBO J., 9:1665 (1990); J. O. Boles, K. Lewinski, M.Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct.Biol., 1:283 (1994); N. Budisa, B. Steipe, P. Demange, C. Eckerskorn, J.Kellermann and R. Huber, Eur. J. Biochem., 230:788 (1995); and, N.Budisa, W. Karnbrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind,L. Moroder and R. Huber, J. Mol. Biol., 270:616 (1997). Methionineanalogs with alkene or alkyne functionalities have also been insertedefficiently, allowing for additional modification of proteins bychemical means. See, e.g., J. C. M. vanHest and D. A. Tirrell, FEBSLett., 428:68 (1998); J. C. M. van Hest, K. L. Kiick and D. A. Tirrell,J. Am. Chem. Soc., 122:1282 (2000); and, K. L. Kiick and D. A. Tirrell,Tetrahedron, 56:9487 (2000).

The success of this method depends on the recognition of the unnaturalamino acid analogs by aminoacyl-tRNA synthetases, which, in general,require high selectivity to insure the fidelity of protein translation.Therefore, the range of chemical functionality accessible via this routeis limited. For instance, although thiaproline can be incorporatedquantitatively into proteins, oxaproline and selenoproline cannot. See,N. Budisa, C. Minks, F. J. Medrano, J. Lutz, R. Huber and L. Moroder,Proc. Natl. Acad. Sci. USA, 95:455 (1998). One way to expand the scopeof this method is to relax the substrate specificity of aminoacyl-tRNAsynthetases, which has been achieved in a limited number of cases. Forexample, it was found that replacement of Ala²⁹⁴ by Gly in Escherichiacoli phenylalanyl-tRNA synthetase (PheRS) increases the size ofsubstrate binding pocket, and results in the acylation of tRNAPhe byp-Cl-phenylalanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke,Biochemistry, 33:7107 (1994). An Escherichia coli strain harboring thismutant PheRS allows the incorporation of p-Cl-phenylalanine orp-Br-phenylalanine in place of phenylalanine. See, e.g., M. Ibba and H.Hennecke, FEBS Lett., 364:272 (1995); and, N. Sharma, R. Furter, P. Kastand D. A. Tirrell, FEBS Lett., 467:37 (2000). Similarly, a pointmutation Phe130Ser near the amino acid binding site of Escherichia colityrosyl-tRNA synthetase was shown to allow azatyrosine to beincorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T.Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soil andS, Nishimura, J. Biol. Chem., 275:40324 (2000).

The fidelity of aminoacylation is maintained both at the level ofsubstrate discrimination and proofreading of non-cognate intermediatesand products. Therefore, an alternative strategy to incorporateunnatural amino acids into proteins in vivo is to modify synthetasesthat have proofreading mechanisms. These synthetases cannot discriminateand therefore activate amino acids that are structurally similar to thecognate natural amino acids. This error is corrected at a separate site,which deacylates the mischarged amino acid from the tRNA to maintain thefidelity of protein translation. If the proofreading activity of thesynthetase is disabled, structural analogs that are misactivated mayescape the editing function and be incorporated. This approach has beendemonstrated recently with the valyl-tRNA synthetase (ValRS). See, V.Doring, H. D. Mootz, L. A. Nangle, T. L. Hendrickson, V. deCrecy-Lagard, P. Schimmel and P. Marliere, Science, 292:501 (2001).ValRS can misaminoacylate tRNAVal with Cys, Thr, or aminobutyrate (Abu);these noncognate amino acids are subsequently hydrolyzed by the editingdomain. After random mutagenesis of the Escherichia coli chromosome, amutant Escherichia coli strain was selected that has a mutation in theediting site of ValRS. This edit-defective ValRS incorrectly chargestRNAVal with Cys. Because Abu sterically resembles Cys (—SH group of Cysis replaced with —CH3 in Abu), the mutant ValRS also incorporates Abuinto proteins when this mutant Escherichia coli strain is grown in thepresence of Abu. Mass spectrometric analysis shows that about 24% ofvalines are replaced by Abu at each valine position in the nativeprotein.

At least one major limitation of the methods described above is that allsites corresponding to a particular natural amino acid throughout theprotein are replaced. The extent of incorporation of the natural andunnatural amino acid may also vary—only in rare cases can quantitativesubstitution be achieved since it is difficult to completely deplete thecognate natural amino acid inside the cell. Another limitation is thatthese strategies make it difficult to study the mutant protein in livingcells, because the multisite incorporation of analogs often results intoxicity. Finally, this method is applicable in general only to closestructural analogs of the common amino acids, again becausesubstitutions must be tolerated at all sites in the genome.

Solid-phase synthesis and semisynthetic methods have also allowed forthe synthesis of a number of small proteins containing novel aminoacids. For example, see the following publications and references citedwithin, which are as follows: Crick, F. J. C., Barrett, L. Brenner, S.Watts-Tobin, R. General nature of the genetic code for proteins. Nature,1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides. XXXVI.The effect of pyrazole-imidazole replacements on the S-proteinactivating potency of an S-peptide fragment, J. Am Chem, 5914-5919(1966); Kaiser, E. T. Synthetic approaches to biologically activepeptides and proteins including enzymes, Acc Chem Res, 47-54 (1989);Nakatsuka, T., Sasaki, T., Kaiser, E. T. Peptide segment couplingcatalyzed by the semisynthetic enzyme thiosubtilisin, J Am Chem Soc,3808-3810 (1987); Schnolzer, M., Kent, S B H. Constructing proteins bydovetailing unprotected synthetic peptides: backbone-engineered HIVprotease, Science, 221-225 (1992); Chaiken, I. M. Semisynthetic peptidesand proteins, CRC Crit Rev Biochem, 255-301 (1981); Offord, R. E.Protein engineering by chemical means? Protein Eng., 151-157 (1987);and, Jackson, D. Y., Burnier, J., Quan, C., Stanley, M., Tom, J., Wells,J. A. A Designed Peptide Ligase for Total Synthesis of Ribonuclease Awith Unnatural Catalytic Residues, Science, 243 (1994).

Chemical modification has been used to introduce a variety of unnaturalside chains, including cofactors, spin labels and oligonucleotides intoproteins in vitro. See, e.g., Corey, D. R., Schultz, P. G. Generation ofa hybrid sequence-specific single-stranded deoxyribonuclease, Science,1401-1403 (1987); Kaiser, E. T., Lawrence D. S., Rokita, S. E. Thechemical modification of enzymatic specificity, Rev Biochem, 565-595(1985); Kaiser, E. T., Lawrence, D. S. Chemical mutation of enzymeactive sites, Science, 505-511 (1984); Neet, K. E., Nanci A, Koshland,D. E. Properties of thiol-subtilisin, J. Biol. Chem., 6392-6401 (1968);Polgar, L. B., M. L. A new enzyme containing a synthetically formedactive site. Thiol-subtilisin. J. Am Chem Soc, 3153-3154 (1966); and,Pollack, S. J., Nakayama, G. Schultz, P. G. Introduction of nucleophilesand spectroscopic probes into antibody combining sites, Science,1038-1040 (1988).

Alternatively, biosynthetic methods that employ chemically modifiedaminoacyl-tRNAs have been used to incorporate several biophysical probesinto proteins synthesized in vitro. See the following publications andreferences cited within: Brunner, J. New Photolabeling and crosslinkingmethods, Annu. Rev Biochem, 483-514 (1993); and, Krieg, U. C., Walter,P., Hohnson, A. E. Photocrosslinking of the signal sequence of nascentpreprolactin of the 54-kilodalton polypeptide of the signal recognitionparticle, Proc. Natl. Acad. Sci, 8604-8608 (1986).

Previously, it has been shown that unnatural amino acids can besite-specifically incorporated into proteins in vitro by the addition ofchemically aminoacylated suppressor tRNAs to protein synthesis reactionsprogrammed with a gene containing a desired amber nonsense mutation.Using these approaches, one can substitute a number of the common twentyamino acids with close structural homologues, e.g., fluorophenylalaninefor phenylalanine, using strains auxotropic for a particular amino acid.See, e.g., Noren, C. J., Anthony-Cahill, Griffith, M. C., Schultz, P. G.A general method for site-specific incorporation of unnatural aminoacids into proteins, Science, 244: 182-188 (1989); M. W. Nowak, et al.,Science 268:439-42 (1995); Bain, J. D., Glabe, C. G., Dix, T. A.,Chamberlin, A. R., Diala, E. S. Biosynthetic site-specific Incorporationof a non-natural amino acid into a polypeptide, J. Am Chem Soc,111:8013-8014 (1989); N. Budisa et al., FASEB J. 13:41-51 (1999);Ellman, J. A., Mendel, D., Anthony-Cahill, S., Noren, C. J., Schultz, P.G. Biosynthetic method for introducing unnatural amino acidssite-specifically into proteins, Methods in Enz., 301-336 (1992); and,Mendel, D., Cornish, V. W. & Schultz, P. G. Site-Directed Mutagenesiswith an Expanded Genetic Code, Annu Rev Biophys. Biomol Struct. 24,435-62 (1995).

For example, a suppressor tRNA was prepared that recognized the stopcodon UAG and was chemically aminoacylated with an unnatural amino acid.Conventional site-directed mutagenesis was used to introduce the stopcodon TAG, at the site of interest in the protein gene. See, e.g.,Sayers, J. R., Schmidt, W. Eckstein, F. 5′, 3′ Exonuclease inphosphorothioate-based olignoucleotide-directed mutagensis, NucleicAcids Res, 791-802 (1988). When the acylated suppressor tRNA and themutant gene were combined in an in vitro transcription/translationsystem, the unnatural amino acid was incorporated in response to the UAGcodon which gave a protein containing that amino acid at the specifiedposition. Experiments using [³H]-Phe and experiments with α-hydroxyacids demonstrated that only the desired amino acid is incorporated atthe position specified by the UAG codon and that this amino acid is notincorporated at any other site in the protein. See, e.g., Noren, et al,supra; and, Ellman, J. A., Mendel, D., Schultz, P. G. Site-specificincorporation of novel backbone structures into proteins, Science,197-200 (1992).

In general, these in vitro approaches are limited by difficulties inachieving site-specific incorporation of the amino acids, by therequirement that the amino acids be simple derivatives of the commontwenty amino acids or problems inherent in the synthesis of largeproteins or peptide fragments.

Microinjection techniques have also been use incorporate unnatural aminoacids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R.Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J.Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty andH. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin.Chem. Biol., 4:645 (2000). A Xenopus oocyte was coinjected with two RNAspecies made in vitro: an mRNA encoding the target protein with a UAGstop codon at the amino acid position of interest and an ambersuppressor tRNA aminoacylated with the desired unnatural amino acid. Thetranslational machinery of the oocyte then inserts the unnatural aminoacid at the position specified by UAG. This method has allowed in vivostructure-function studies of integral membrane proteins, which aregenerally not amenable to in vitro expression systems. Examples includethe incorporation of a fluorescent amino acid into tachykininneurokinin-2 receptor to measure distances by fluorescence resonanceenergy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U.Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J.Biol. Chem., 271:19991 (1996); the incorporation of biotinylated aminoacids to identify surface-exposed residues in ion channels, see, e.g.,J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739(1997); the use of caged tyrosine analogs to monitor conformationalchanges in an ion channel in real time, see, e.g., J. C. Miller, S. K.Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron,20:619 (1998); and, the use of alpha hydroxy amino acids to change ionchannel backbones for probing their gating mechanisms. See, e.g., P. M.England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999);and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J.Yang, Nat. Neurosci., 4:239 (2001).

However, there are limitations microinjection method, e.g., thesuppressor tRNA has to be chemically aminoacylated with the unnaturalamino acid in vitro, and the acylated tRNA is consumed as astoichiometric reagent during translation and cannot be regenerated.This limitation results in poor suppression efficiency and low proteinyields, necessitating highly sensitive techniques to assay the mutantprotein such as electrophysiological measurements. Moreover, this methodis only applicable to cells that can be microinjected.

The ability to incorporate unnatural amino acids directly into proteinsin vivo offers the advantages of high yields of mutant proteins,technical ease, the potential to study the mutant proteins in cells orpossibly in living organisms and the use of these mutant proteins intherapeutic treatments. The ability to include unnatural amino acidswith various sizes, acidities, nucleophilicities, hydrophobicities, andother properties into proteins can greatly expand our ability torationally and systematically manipulate the structures of proteins,both to probe protein function and create new proteins or organisms withnovel properties. However, the process is difficult, because the complexnature of tRNA-synthetase interactions that are required to achieve ahigh degree of fidelity in protein translation.

In one attempt to site-specifically incorporate para-F-Phe, a yeastamber suppressor tRNAPheCUA/phenylalanyl-tRNA synthetase pair was usedin a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See,e.g., R. Furter, Protein Sci., 7:419 (1998). Because yeast PheRS doesnot have high substrate specificity for p-F-Phe, the mutagenesis sitewas translated with only 64-75% p-F-Phe and the remainder as Phe and Lyseven in the excess of p-F-Phe added to the growth media. In addition, atthe Phe codon positions, 7% p-F-Phe was found, indicating that theendogenous Escherichia coli PheRS incorporates p-F-Phe in addition toPhe. Besides of its translational infidelity, e.g., the suppressor tRNAand PheRS are not truly orthogonal, this approach is not generallyapplicable to other unnatural amino acids.

Therefore, improvements to the process are needed to provide moreefficient and effective methods to alter the biosynthetic machinery ofthe cell. The present invention addresses these and other needs, as willbe apparent upon review of the following disclosure.

SUMMARY OF THE INVENTION

The present invention provides a variety of methods for making and usingtranslation systems that can incorporate unnatural amino acids intoproteins, as well as related compositions. Proteins comprising unnaturalamino acids made by the translation system are also a feature of theinvention. Both known and new unnatural amino acids can be incorporatedinto proteins using the translation system of the invention. Theinvention further provides novel unnatural amino acids; variouscompositions including the unnatural amino acids, e.g., proteins andcells including unnatural amino acids; chemical and biosynthetic methodsfor producing unnatural amino acids; and methods for producing andcompositions comprising an autonomous twenty-one amino acid cell.

Thus, in one aspect, the present invention provides compositionscomprising a translation system. The translation system comprises anorthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase(O-RS). Typically, the O-RS preferentially aminoacylates the O-tRNA withat least one unnatural amino acid in the translation system and theO-tRNA recognizes at least one selector codon. The translation systemthus inserts the unnatural amino acid into a protein produced in thesystem, in response to an encoded selector codon.

Typical translation systems include cells, such as bacterial cells(e.g., Escherichia coli), archeaebacterial cells, eukaryotic cells(e.g., yeast cells, mammalian cells, plant cells, insect cells), or thelike. Alternatively, the translation system comprises an in vitrotranslation system, e.g., a translation extract including a cellularextract.

Example O-tRNAs comprise a nucleic acid comprising a polynucleotidesequence selected from the group consisting of: SEQ ID NO:1-3 and/or acomplementary polynucleotide sequence thereof. Similarly, example O-RSinclude polypeptides selected from the group consisting of: apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 35-66 and a polypeptide encoded by a nucleicacid comprising a polynucleotide sequence selected from the groupconsisting of: SEQ ID NO: 4-34 and a complementary polynucleotidesequence thereof.

Examples of unnatural amino acids that can be used by the translationsystem include: an unnatural analogue of a tyrosine amino acid; anunnatural analogue of a glutamine amino acid; an unnatural analogue of aphenylalanine amino acid; an unnatural analogue of a serine amino acid;an unnatural analogue of a threonine amino acid; an alkyl, aryl, acyl,azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynl,ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate,phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde,hydroxylamine, keto, or amino substituted amino acid, or any combinationthereof; an amino acid with a photoactivatable cross-linker; aspin-labeled amino acid; a fluorescent amino acid; an amino acid with anovel functional group; an amino acid that covalently or noncovalentlyinteracts with another molecule; a metal binding amino acid; ametal-containing amino acid; a radioactive amino acid; a photocagedand/or photoisomerizable amino acid; a biotin or biotin-analoguecontaining amino acid; a glycosylated or carbohydrate modified aminoacid; a keto containing amino acid; amino acids comprising polyethyleneglycol or polyether; a heavy atom substituted amino acid; a chemicallycleavable or photocleavable amino acid; an amino acid with an elongatedside chain; an amino acid containing a toxic group; a sugar substitutedamino acid, e.g., a sugar substituted serine or the like; acarbon-linked sugar-containing amino acid; a redox-active amino acid; anα-hydroxy containing acid; an amino thio acid containing amino acid; anα,α disubstituted amino acid; a β-amino acid; and a cyclic amino acidother than proline.

For example, the unnatural amino acid can be an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine in one embodiment, the at least one unnaturalamino acid is an O-methyl-L-tyrosine. In one specific exampleembodiment, the at least one unnatural amino acid is anL-3-(2-naphthyl)alanine. In another set of specific examples, the atleast one unnatural amino acid is an amino-, isopropyl-, orO-allyl-containing phenylalanine analogue.

Any of a variety of selector codons can be used in the presentinvention, including nonsense codons, rare codons, four (or more) basecodons, or the like. For example, in one embodiment, the at least oneselector codon is an amber codon.

A variety of exemplar translation systems are provided herein, includinge.g., an Escherichia coli cell comprising a mtRNA_(CUA) ^(Tyr) and amutant TyrRS (LWJ16), where the mutant TyrRS (LWJ16) preferentiallyaminoacylates the mtRNA_(CUA) ^(Tyr) with O-methyl-L-tyrosine in thecell and the cell uses the mtRNA_(CUA) ^(Tyr) to recognize an ambercodon. In another example, an Escherichia coli cell comprising amtRNA_(CUA) ^(Tyr) and an SS12-TyrRS is provided, where the SS12-TyrRSpreferentially aminoacylates the mtRNA_(CUA) ^(Tyr) withL-3-(2-naphthyl)alanine in the cell and the cell uses the mtRNA_(CUA)^(Tyr) to recognize an amber codon.

The translation system herein provides the ability to synthesizeproteins that comprise unnatural amino acids in usefully largequantities. For example, proteins comprising at least one unnaturalamino acid can be produced at a concentration of at least about 10, 50,100 or more micrograms per liter, e.g., in a composition comprising acell extract, a buffer, a pharmaceutically acceptable excipient, and/orthe like.

Another aspect of the present invention provides for the production ofproteins that are homologous to any available protein, but comprisingone or more unnatural amino acid homologue. For example, therapeuticproteins can be made that comprise one or more unnatural amino acid andare homologous to one or more therapeutic protein. For example, in oneaspect, the protein is homologous to a therapeutic or other protein suchas: a cytokine, a growth factor, a growth factor receptor, aninterferon, an interleukin, an inflammatory molecule, an oncogeneproduct, a peptide hormone, a signal transduction molecule, a steroidhormone receptor, a transcriptional activator, a transcriptionalsuppressor, erythropoietin (EPO), insulin, human growth hormone,epithelial Neutrophil Activating Peptide-78, GROα/MGSA, GROβ, GROγ,MIP-1α, MIP-1β, MCP-1, hepatocyte growth factor, insulin-like growthfactor, leukemia inhibitory factor, oncostatin M, PD-ECSF, PDGF,pleiotropin, SCF, c-kit ligand, VEGEF, G-CSF, IL-1, IL-2, IL-8, IGF-I,IGF-II, FGF (fibroblast growth factor), PDGF, TNF, TGF-α, TGF-β, EGF(epidermal growth factor), KGF (keratinocyte growth factor), SCF/c-Kit,CD40UCD40, VLA-4/VCAM-1, ICAM-1/LFA-1, hyalurin/CD44, Mos, Ras, Raf,Met; p53, Tat, Fos, Myc, Jun, Myb, Rel, estrogen receptor, progesteronereceptor, testosterone receptor, aldosterone receptor, LDL receptor,and/or corticosterone. In another set of embodiments, the protein ishomologous to a therapeutic or other protein such as: an Alpha-1antitrypsin, an Angiostatin, an Antihemolytic factor, an antibody, anApolipoprotein, an Apoprotein, an Atrial natriuretic factor, an Atrialnatriuretic polypeptide, an Atrial peptide, a C—X—C chemokine, T39765,NAP-2, ENA-78, a Gro-a, a Gro-b, a Gro-c, an IP-10, a GCP-2, an NAP-4,an SDF-1, a PF4, a MIG, a Calcitonin, a c-kit ligand, a cytokine, a CCchemokine, a Monocyte chemoattractant protein-1, a Monocytechemoattractant protein-2, a Monocyte chemoattractant protein-3, aMonocyte inflammatory protein-1 alpha, a Monocyte inflammatory protein-1beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, aCD40, a CD40 ligand, a C-kit Ligand, a Collagen, a Colony stimulatingfactor (CSF), a Complement factor 5a, a Complement inhibitor, aComplement receptor 1, a cytokine, an epithelial Neutrophil ActivatingPeptide-78, a GROα/MGSA, a GROβ, a GROγ, a MIP-1α, a MIP-1β, a MCP-1, anEpidermal Growth Factor (EGF), an epithelial Neutrophil ActivatingPeptide, an Erythropoietin (EPO), an Exfoliating toxin, a Factor IX, aFactor VII, a Factor VIII, a Factor X, a Fibroblast Growth Factor (FGF),a Fibrinogen, a Fibronectin, a G-CSF, a GM-CSF, a Glucocerebrosidase, aGonadotropin, a growth factor, a growth factor receptor, a Hedgehogprotein, a Hemoglobin, a Hepatocyte Growth Factor (HGF), a Hirudin, aHuman serum albumin, an ICAM-1, an ICAM-1 receptor, an LFA-1, an LFA-1receptor, an Insulin, an Insulin-like Growth Factor (IGF), an IGF-I, anIGF-II, an interferon, an IFN-α, an IFN-β, an IFN-γ, an interleukin, anIL-1, an IL-2, an IL-3, an IL-4, an IL-5, an IL-6, an IL-7, an IL-8, anIL-9, an IL-10, an IL-11, an IL-12, a Keratinocyte Growth Factor (KGF),a Lactoferrin, a leukemia inhibitory factor, a Luciferase, a Neurturin,a Neutrophil inhibitory factor (NIF), an oncostatin M, an Osteogenicprotein, an oncogene product, a Parathyroid hormone, a PD-ECSF, a PDGF,a peptide hormone, a Human Growth Hormone, a Pleiotropin, a Protein A, aProtein G, a Pyrogenic exotoxins A, B, or C, a Relaxin, a Renin, an SCF,a Soluble complement receptor I, a Soluble I-CAM 1, a Solubleinterleukin receptors, a Soluble TNF receptor, a Somatomedin, aSomatostatin, a Somatotropin, a Streptokinase, a Superantigens, aStaphylococcal enterotoxins, an SEA, an SEB, an SEC1, an SEC2, an SEC3,an SED, an SEE, a steroid hormone receptor, a Superoxide dismutase, aToxic shock syndrome toxin, a Thymosin alpha 1, a Tissue plasminogenactivator, a tumor growth factor (TGF), a TGF-α, a TGF-β, a TumorNecrosis Factor, a Tumor Necrosis Factor alpha, a Tumor necrosis factorbeta, a Tumor necrosis factor receptor (TNFR), a VLA-4 protein, a VCAM-1protein, aVascular Endothelial Growth Factor (VEGEF), a Urokinase, aMos, a Ras, a Raf, a Met; a p53, a Tat, a Fos, a Myc, a Jun, a Myb, aRel, an estrogen receptor, a progesterone receptor, a testosteronereceptor, an aldosterone receptor, an LDL receptor, and/or acorticosterone. In one aspect, the compositions herein comprise aprotein comprising an unnatural amino acid and a pharmaceuticallyacceptable excipient, including, e.g., any of the proteins noted aboveand a pharmaceutically acceptable excipient.

Homology to the polypeptide can be inferred by performing a sequencealignment, e.g., using BLASTN or BLASTP, e.g., set to defaultparameters. For example, in one embodiment, the protein is at leastabout 50%, at least about 75%, at least about 80%, at least about 90% orat least about 95% identical to a known therapeutic protein (e.g., aprotein present in Genebank or other available databases). For example,in one preferred embodiment, the therapeutic protein is erythropoietin(EPO).

The protein of interest can contain 1, 2, 3, 4, 5, 6, 7, 6, 9, 10, 11,12, 13, 14, 15 or more unnatural amino acids. The unnatural amino acidscan be the same or different, e.g., there can be 1, 2, 3, 4, 5, 6, 7, 6,9, 10, 11, 12, 13, 14, 15 or more different sites in the protein thatcomprise 1, 2, 3, 4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14, 15 or moredifferent unnatural amino acids. For example, in one embodiment, theprotein is DHFR, and the at least one unnatural amino acid is selectedfrom the group consisting of O-methyl-L-tyrosine andL-3-(2-naphthyl)alanine.

The present invention also provides methods for producing at least oneprotein in a translation system such that the at least one proteincomprises at least one unnatural amino acid. In the methods, thetranslation system is provided with at least one nucleic acid comprisingat least one selector codon, wherein the nucleic acid encodes the atleast one protein. The translation system is also provided with anorthogonal tRNA (O-tRNA), that functions in the translation system andrecognizes the at least one selector codon and an orthogonal aminoacyltRNA synthetase (O-RS), that preferentially aminoacylates the O-tRNAwith the at least one unnatural amino acid in the translation system.The translation system is also provided with the at least one unnaturalamino, thereby producing, in the translation system, the at least oneprotein comprising the at least one unnatural amino acid.

All of the above structural features of the compositions can be embodiedin the methods, e.g., types of translation systems (e.g., cells, cellextracts, etc.), types of proteins produced in the translation systems(e.g., EPO homologues and the other proteins noted herein) specificmutant proteins, specific unnatural amino acids, and the like.

In one aspect, the protein(s) comprising unnatural amino acids that areproduced are processed and modified in a cell-dependent manner. Thisprovides for the production of proteins that are stably folded,glycosylated, or otherwise modified by the cell.

The unnatural amino acid is optionally provided exogenously to thetranslation system. Alternately, e.g., where the translation system is acell, the unnatural amino acid can be biosynthesized by the translationsystem.

In one specific example embodiment, the invention provides methods forproducing in an Escherichia coli cell at least one protein comprising atleast one O-methyl-L-tyrosine. The method includes providing thetranslation system with at least one nucleic acid comprising an ambercodon, wherein the nucleic acid encodes the at least one protein;providing the translation system with a mtRNA_(CUA) ^(Tyr), wherein themtRNA_(CUA) ^(Tyr) functions in the cell and wherein the mtRNA_(CUA)^(Tyr) recognizes the amber codon; providing the translation system witha mutant TyrRS (LWJ16), wherein the mutant TyrRS (LWJ16) aminoacylatesthe mtRNA_(CUA) ^(Tyr) with the O-methyl-L-tyrosine in the cell; and,providing the cell with the O-methyl-L-tyrosine, thereby producing inthe cell at least one protein comprising the O-methyl-L-tyrosine.

In another example embodiment, the invention provides a method forproducing in an Escherichia coli cell at least one protein comprising atleast one L-3-(2-naphthyl)alanine. In this example embodiment, themethod includes: providing the translation system with at least onenucleic acid comprising an amber codon, wherein the nucleic acid encodesthe at least one protein; providing the cell with a mtRNA_(CUA) ^(Tyr),wherein the mtRNA_(CUA) ^(Tyr) functions in the cell and wherein themtRNA_(CUA) ^(Tyr) recognizes the amber codon; providing the cell withan SS12-TyrRS, wherein the SS12-TyrRS aminoacylates the mtRNA_(CUA)^(Tyr) with the L-3-(2-naphthyl)alanine in the cell; and, providing thecell with the L-3-(2-naphthyl)alanine, thereby producing in the cell atleast one protein comprising the L-3-(2-naphthyl)alanine.

In another aspect, the present invention provides unnatural amino acids,e.g., meta substituted phenylalanine analogues, such as3-acetyl-phenylalanine and 3-methoxy phenylalanine; tyrosine analogues,such as 4-allyl tyrosine; glycosylated amino acids, and the like.

Various compositions comprising unnatural amino acids, e.g., proteinsand cells comprising the unnatural amino acids of the invention, arealso provided. For example, compositions comprising an unnatural aminoacid and an orthogonal tRNA, e.g., covalently bonded, are provided.Compositions comprising unnatural amino acids and an orthogonalaminoacyl tRNA synthetase, e.g., hydrogen bonded, are also provided.

In another aspect, the present invention provides methods ofsynthesizing amino acids. For example, 4-allyl-L-tyrosine, is typicallysynthesized by reacting a protected tyrosine with allyl bromide, e.g.,in the presence of sodium hydride and DMF, and deprotecting to yield4-allyl-L-tyrosine. Typically an NBoc or Fmoc protected tyrosine isused, e.g., with an acidic deprotection, e.g., in the presence ofhydrochloric acid and dioxane. The final product is optionallyextracted, e.g., with ethanol or dichloromethane.

Meta-substituted phenylalanine analogues are typically synthesized bycondensing diethylacetamidomalonate and a meta-substituted benzylbromide. The product of the condensation is then typically hydrolyzed toyield the meta-substituted phenylalanine analogue, e.g., a keto, acetyl,or methoxy substituted phenylalanine such as 3-methoxy-phenylalanine or3-acetyl-phenylalanine. The desired meta substituted benzyl bromide isoptionally synthesized by reacting N-bromosuccinimide (NBS) with3-methylacetophenone to produce a brominated product, and crystallizingthe brominated product in a hexane solution. The crystallization yieldsa monobromide product as opposed to a mixture of a monobromide and adibromide.

In another aspect, the present invention provides biosynthetic methodsfor producing unnatural amino acids. For example, glycosylated aminoacids are optionally synthesized in vivo, e.g., by transforming a cellwith a plasmid comprising a gene for an N-acetyl-galactosaminidase, atransglycosylase, or a serine-glycosylhydrolase. The cell then producesthe desired glycosylated amino acid, e.g. from cellular resources. Inanother example, p-aminophenylalanine is synthesized, e.g., in vivo, byenzymatically converting chorismate to 4-amino-4-deoxychorismic acid;which is enzymatically converted to 4-amino-4-deoxyprephenic acid; andenzymatically converting the 4-amino-4-deoxyprephenic acid top-aminophenyl-pyruvic acid, which is enzymatically converted top-aminophenylalanine. The enzymatic conversions are typically performedusing a 4-amino-4-deoxychorismate synthase, e.g., PapA, a chorismatemutase, e.g., Pap B, and a prephenate dehydrogenase, e.g., PapC,respectively. The final step is typically performed by contacting thep-aminophenyl-pyruvic acid with an aminotransferease, e.g., anon-specific tyrosine aminotransferase, e.g., derived from E coli.Aminotransfereases of use in the present invention include, but are notlimited to, tyrB, aspS, or ilvE. Typically the above steps are performedin vivo, e.g., by transforming a cell with a plasmid comprising thegenes which encode the enzymes used for the synthesis.

In another aspect, the present invention provides a method of producingp-aminophenylalanine in an Escherichia coli cell. The method typicallycomprises transforming the cell with a plasmid comprising papA, papB,and papC, wherein the cell comprises chorismate and an aminotransferase.Expression of papA, papB, and papC results in a synthase, a mutase, anda dehydrogenase, wherein these enzymes together with theaminotransferase produce p-phenylalanine from chorismate.

In another aspect, the present invention provides an autonomoustwenty-one (or more) amino acid cell. The cell, e.g., a bacterial cell,typically comprises a biosynthetic pathway system for producing anunnatural amino acid, e.g., p-aminophenylalanine, from one or morecarbon sources within the cell, e.g., chorismate, and a translationsystem comprising an orthogonal tRNA (O-tRNA) and an orthogonalaminoacyl tRNA synthetase (O-RS). The O-RS preferentially aminoacylatesthe O-tRNA with the unnatural amino acid and the O-tRNA incorporates theunnatural amino acid into a protein in response to a selector codon,e.g., a nonsense codon such as TAG, a four base codon, or an ambercodon. The cell can comprise more than one unnatural amino acid, e.g. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more unnatural aminoacids, optionally with more than one orthogonal tRNA (e.g., one perunnatural amino acid to provide for site-specific incorporation of eachunnatural amino acid in a protein, or more, or less, to tune thespecificity of unnatural amino acid incorporation) and/or more than oneorthogonal aminoacyl tRNA synthetase (O-RS) (e.g., one per orthogonaltRNA, or more or less to tune the specificity of unnatural amino acidincorporation).

In some embodiments, the biosynthetic pathway systems produce a naturalcellular amount of the unnatural amino acid, e.g., the cell produces theunnatural amino acid in an amount sufficient for protein biosynthesis,which amount does not substantially alter the concentration of naturalamino acids or substantially exhaust cellular resources in theproduction of the unnatural amino acids.

In one example class of embodiments, the autonomous cell is engineeredto produce p-aminophenylalanine from chorismate as described above. Inthis embodiment, the cell is engineered to produce the desired enzymesas described above, e.g., a synthase, a dehydrogenase, and a mutasederived from Streptomyces Venezuelae or Streptomyces pristinaespiralisand a aminotransferase derived from E. coli. For example, the cells ofthe invention are optionally transformed with a plasmid, e.g., low copypSC101 derived plasmid, comprising papA, papB, and papC, wherein theplasmid further comprises an lpp promoter and a lac promoter. In someembodiments, the plasmid further comprises one or more ribosome bindingsites.

Other unnatural amino acids that are optionally produced by the cells ofthe invention include, but are not limited to, dopa,O-methyl-L-tyrosine, glycosylated amino acids, pegylated amino acids,other unnatural amino acids noted herein, and the like.

In another related aspect, the present invention provides a cellcomprising one or more systems for producing at least twenty one aminoacids and specifically incorporating one or more of the amino acids intoone or more proteins within the cell, wherein at least one of theincorporated amino acids comprises an unnatural amino acid.

In another aspect, the present invention provides a method ofidentifying an advantage provided by an unnatural amino acid which hasbeen incorporated into one or more proteins of a cell. The methodtypically comprises providing a library of cells, each of which cellscomprises a randomized plasmid, e.g., derived from an E. coli genome.One or more of the randomized plasmids typically confers on the cells anability to incorporate an unnatural amino acid into a protein. Thelibrary of cells is then screened to identify cells with enhancedgrowth, e.g., as compared to a native E. coli cell, thereby identifyingan advantage provided by the unnatural amino acid. In some embodiments,a second screen is used to further verify that any advantage identifiedis due to the unnatural amino acid.

Kits are an additional feature of the invention. For example, the kitscan include one or more translation system as noted above (e.g., a cell,a 21 or more amino acid cell, etc.), one or more unnatural amino acid,e.g., with appropriate packaging material, containers for holding thecomponents of the kit, instructional materials for practicing themethods herein and/or the like. Similarly, products of the translationsystems (e.g., proteins such as EPO analogues comprising unnatural aminoacids) can be provided in kit form, e.g., with containers for holdingthe components of the kit, instructional materials for practicing themethods herein and/or the like.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a sterioview of the amino acid residues in the active site ofTyrRS (modified from P. Brick, T. N. Bhat, D. M. Blow, J. Mol. Biol.208, 83-98 (1988)). Residues from B. stearothermophilus TyrRS are shownin the figure.

FIGS. 2A-2B illustrates accumulation of E. coli DHFR protein, bothwild-type (wt) and mutant under different conditions. Expressionconditions are notated at the top of each lane. The left lane ismolecular weight marker. FIG. 2A is a silver-stained SDS-PAGE gel ofpurified DHFR. FIG. 2B is a Western blot of the gel in FIG. 2A.

FIG. 3 is a tandem mass spectrum of an NH2 terminal peptide of DHFR,MIY*MIAALAVDR (SEQ ID NO:77). The partial sequence Y*MIAALAVDR (aminoacids of 3-12 of SEQ ID NO:77) of the peptide containing theO-methyl-L-tyrosine residue (Y*) can be read from the annotated b or yion series.

FIGS. 4A-4B illustrates accumulation of mouse DHFR protein, bothwild-type (wt) and mutant, under different conditions. Expressionconditions are notated at the top of each lane. The left lane ismolecular weight marker. FIG. 4A is a silver-stained SDS-PAGE gel ofpurified DHFR. FIG. 4B is a Western blot of the gel in FIG. 4A.

FIG. 5 is a tandem mass spectrum of the tryptic peptideLLPEX*TGVLSEVQEEK (SEQ ID NO; 78, X* representsL-3-(2-naphthyl)-alanine). The sequence can be read from the annotated bor y ion series; even so, b7 and y13 are not observed. The base peak821.7 (100%) assigned to the doubly charged y14 ion is truncated forclarity.

FIG. 6, Panels A-D, illustrate features of the amplifiable fluorescencereporter system. FIG. 6A is plasmid pREP. T7 RNA polymerasetranscription is controlled by the ara promoter; protein expressiondepends on suppression of amber codons at varying locations within thegene. GFPuv expression is controlled by T7 RNA polymerase. Plasmid pREPis compatible for use with a ColE1 plasmid expressing an orthogonalsynthetase/tRNA pair. FIG. 6B illustrates composition and fluorescenceenhancement of T7 RNA polymerase gene constructs within pREP(1-12). Theconstruct number is indicated to the left of each. Fluorescenceenhancements, indicated to the right of each construct, are calculatedas the cell concentration-corrected ratio of fluorescence, as measuredfluorimetrically, of cells containing pREP(1-12) and pQ or pQD. Thepositions of amber mutations within the gene are indicated above eachconstruct. FIG. 6C illustrates cytometric analysis of cells containingpREP(10) and either pQD (top) or pQ (bottom). FIG. 6D illustratesfluorimetric analyses of cells containing pREP(10) and expressingvarious E. coli suppressor tRNAs. ‘None’ indicates that the cellscontain no suppressor tRNA.

FIG. 7, Panels A-C, illustrates components of a multipurpose reporterplasmid system for directing the evolution of M. jannaschii TyrRS. FIG.7A illustrates plasmid pREP/YC-JYCUA. Plasmid pREP/YC-JYCUA iscompatible for use with plasmid pBK and variants. FIG. 7B illustratesstructures of unnatural amino acids used as targets for the evolution ofM. jannaschii TyrRS. FIG. 7C illustrates a strategy for a evolution ofan aminoacyl-tRNA synthetase using plasmid pREP/YC-JYCUA. Fluorescentand non-fluorescent cells are shown in black and grey, respectively.

FIG. 8, Panels A-D, illustrates the activity of the dominant synthetasevariant from each successful evolution experiment. FIG. 8A is aphotograph illustrating long-wavelength ultraviolet illumination ofcells containing pREP/YC-JYCUA and the indicated synthetase variant,grown in either the presence (+) or absence (−) of the correspondingunnatural amino acid. FIG. 8B illustrates a fluorimetric analysis ofcells containing pREP/YC-JYCUA and the indicated synthetase variant,grown in either the presence (left) or absence (right) of thecorresponding unnatural amino acid. FIG. 8C is a table that illustratesa Cm IC₅₀ analysis of cells containing pREP/YC-JYCUA and the indicatedsynthetase variant, grown in either the presence or absence of thecorresponding unnatural amino acid. FIG. 8D illustrates a proteinexpression analysis from cells containing pBAD/JYAMB-4TAG and theindicated synthetase variant, grown in either the presence (+) orabsence (−) of the corresponding unnatural amino acid.

FIG. 9 illustrates activity comparisons of OAY-RS variants derived usinga negative FACS-based screen [OAY-RS(1,3,5)] or negative barnase-basedselection [OAY-RS(B)]. Cells containing pREP/YC-JYCUA and the indicatedsynthetase variant were grown in either the presence (solid block, left)or absence (solid block, right) of the corresponding unnatural aminoacid and analyzed fluorimetrically. Fluorescence enhancement (bar, back)is calculated as the cell concentration-corrected ratio of fluorescenceof cells grown in the presence versus the absence of unnatural aminoacid.

FIG. 10 is an autoradiograph of a western blot demonstrating expressionof m-MeO-Phe- and m-Acetyl-Phe-incorporated DHFR.

FIG. 11 illustrates the fluorescence emission spectra of fluoresceinhydrazide labelled protein.

FIG. 12 illustrates the unnatural amino acids para-azido-phenylalanineand para-benzoyl-phenylalanine.

FIG. 13 illustrates a chemical scheme for the synthesis of anallyl-substituted phenylalanine.

FIG. 14 illustrates a chemical scheme for the synthesis ofmeta-substituted phenylalanines.

FIGS. 15A-15B illustrates the biosynthesis of p-aminophenylalanine. FIG.15A illustrates a plasmid used for the biosynthesis ofp-aminophenylalanine and FIG. 15B illustrates a biosynthetic scheme forthe production of p-aminophenylalanine from chorismate, e.g., using theplasmid of FIG. 15A.

FIG. 16 illustrates a variety of unnatural amino acids.

FIG. 17 illustrates a variety of unnatural amino acids.

FIG. 18 illustrates a variety of unnatural amino acids.

FIG. 19 illustrates additional amino acids, natural and unnatural forincorporation into proteins via in vivo suppression.

FIG. 20 provides a biosynthetic scheme for production of dopa.

FIG. 21 illustrates a method for determining evolutionary advantages ina cell due to the ability to specifically incorporate twenty-one aminoacids.

FIG. 22 illustrates a method for site-specific incorporation ofunnatural amino acids.

FIG. 23 illustrates the synthesis of various glutamine analogs.

FIG. 24 illustrates the synthesis of a gamma substituted glutamineanalog.

FIG. 25 illustrates the synthesis of a cyclic glutamine derivative.

FIG. 26 illustrates a variety of tyrosine analogs.

FIG. 27 illustrates a synthetic scheme for the production of tyrosineanalogs.

FIG. 28 illustrates a biosynthetic scheme for producing glycosylatedamino acids.

FIG. 29 illustrates a variety of unnatural amino acids, e.g., as used ina cellular uptake study. Any or all of the above figures are schematicin nature.

DETAILED DESCRIPTION In General

The present invention provides compositions and methods for augmentingthe protein biosynthetic machinery of a cell to accommodate additionalgenetically encoded amino acids using orthogonal tRNA/aminoacyl tRNAsynthetase (O-tRNA/O-RS) pairs. The compositions and methods describedhere can be used with unnatural amino acids, e.g., providing novelspectroscopic, chemical or structural properties to proteins using anyof a wide array of side chains. The invention is applicable to bothprokaryotic (e.g., Eubacteria, Archeaebacteria) and eukaryotic (e.g.,yeast, mammalian, plant, or insect) cells. These compositions andmethods are useful for the site specific incorporation of unnaturalamino acids via selector codons, e.g., stop codons, four base codons,and the like. The invention also provides proteins, including unnaturalamino acids, produced using the compositions or made by the methods ofthe invention. The ability to introduce unnatural amino acids intoproteins directly in living cells provides new tools for studies ofprotein and cellular function and can lead to the generation of proteinswith enhanced properties useful for, e.g., therapeutics.

DEFINITIONS

Homologous: Proteins and/or protein sequences are “homologous” when theyare derived, naturally or artificially, from a common ancestral proteinor protein sequence. Similarly, nucleic acids and/or nucleic acidsequences are homologous when they are derived, naturally orartificially, from a common ancestral nucleic acid or nucleic acidsequence. For example, any naturally occurring nucleic acid can bemodified by any available mutagenesis method to include one or moreselector codon. When expressed, this mutagenized nucleic acid encodes apolypeptide comprising one or more unnatural amino acid. The mutationprocess can, of course, additionally alter one or more standard codon,thereby changing one or more standard amino acid in the resulting mutantprotein as well. Homology is generally inferred from sequence similaritybetween two or more nucleic acids or proteins (or sequences thereof).The precise percentage of similarity between sequences that is useful inestablishing homology varies with the nucleic acid and protein at issue,but as little as 25% sequence similarity is routinely used to establishhomology. Higher levels of sequence similarity, e.g., 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 99% or more can also be used to establishhomology. Methods for determining sequence similarity percentages (e.g.,BLASTP and BLASTN using default parameters) are described herein and aregenerally available.

Orthogonal: As used herein, the term “orthogonal” refers to a molecule(e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNAsynthetase (O-RS)) that is used with reduced efficiency by a system ofinterest (e.g., a translational system, e.g., a cell). Orthogonal refersto the inability or reduced efficiency, e.g., less than 20% efficient,less than 10% efficient, less than 5% efficient, or e.g., less than 1%efficient, of an orthogonal tRNA and/or orthogonal RS to function in thetranslation system of interest. For example, an orthogonal tRNA in atranslation system of interest aminoacylates any endogenous RS of atranslation system of interest with reduced or even zero efficiency,when compared to aminoacylation of an endogenous tRNA by the endogenousRS. In another example, an orthogonal RS aminoacylates any endogenoustRNA in the translation system of interest with reduced or even zeroefficiency, as compared to aminoacylation of the endogenous tRNA by anendogenous RS.

Preferentially aminoacylates: The term “preferentially aminoacylates”refers to an efficiency of, e.g., about 70% efficient, about 75%efficient, about 85% efficient, about 90% efficient, about 95%efficient, or about 99% or more efficient, at which an O-RSaminoacylates an O-tRNA with an unnatural amino acid compared to anaturally occurring tRNA or starting material used to generate theO-tRNA. The unnatural amino acid is then incorporated into a growingpolypeptide chain with high fidelity, e.g., at greater than about 75%efficiency for a given selector codon, at greater than about 80%efficiency for a given selector codon, at greater than about 90%efficiency for a given selector codon, at greater than about 95%efficiency for a given selector codon, or at greater than about 99% ormore efficiency for a given selector codon.

Selector codon: The term “selector codon” refers to codons recognized bythe O-tRNA in the translation process and not recognized by anendogenous tRNA. The O-tRNA anticodon loop recognizes the selector codonon the mRNA and incorporates its amino acid, e.g., an unnatural aminoacid, at this site in the polypeptide. Selector codons can include,e.g., nonsense codons, such as, stop codons, e.g., amber, ochre, andopal codons; four or more base codons; codons derived from natural orunnatural base pairs and the like. For a given system, a selector codoncan also include one of the natural three base codons, wherein theendogenous system does not use said natural three base codon, e.g., asystem that is lacking a tRNA that recognizes the natural three basecodon or a system wherein the natural three base codon is a rare codon.

Suppressor tRNA: A suppressor tRNA is a tRNA that alters the reading ofa messenger RNA (mRNA) in a given translation system. A suppressor tRNAcan read through, e.g., a stop codon, a four base codon, or a rarecodon.

Translation system: The term “translation system” refers to thecomponents necessary to incorporate a naturally occurring amino acidinto a growing polypeptide chain (protein). Components of a translationsystem can include, e.g., ribosomes, tRNAs, synthetases, mRNA and thelike. The components of the present invention can be added to atranslation system, in vivo or in vitro. A translation system can be acell, either prokaryotic, e.g., an E. coli cell, or eukaryotic, e.g., ayeast, mammalian, plant, or insect cell.

Unnatural amino acid: As used herein, the term “unnatural amino acid”refers to any amino acid, modified amino acid, and/or amino acidanalogue that is not one of the 20 naturally occurring amino acids orseleno cysteine.

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein have thesame meaning as commonly understood by those of ordinary skill in theart to which the invention belongs.

Discussion

Proteins are at the crossroads of virtually every biological process,from photosynthesis and vision to signal transduction and the immuneresponse. These complex functions result from a polyamide based polymerconsisting of twenty relatively simple building blocks arranged in adefined primary sequence.

The present invention includes methods and composition for use in thesite-specific incorporation of unnatural amino acids directly intoproteins in vivo. Importantly, the unnatural amino acid is added to thegenetic repertoire, rather than substituting for one of the common 20amino acids. The present invention, e.g., (i) allows the site-selectiveor random insertion of one or more unnatural amino acids at any desiredposition of any protein, (ii) is applicable to both prokaryotic andeukaryotic cells, (iii) enables in vivo studies of mutant proteins inaddition to the generation of large quantities of purified mutantproteins, and (iv) is adaptable to incorporate any of a large variety ofnon-natural amino acids into proteins in vivo. The invention providescompositions and methods useful for in vivo site specific incorporationof unnatural amino acids. Specifically, the invention providestranslation systems, e.g., cells, that include an orthogonal tRNA(O-tRNA), an orthogonal aminoacyl tRNA synthetase (O-RS), and anunnatural amino acid, where the O-RS aminoacylates the O-tRNA with theunnatural amino acid, and the cell uses the components to incorporatethe unnatural amino acid into a growing polypeptide chain.

The invention further provides methods for in vivo site-specificincorporation of unnatural amino acids using the translation systems ofthe invention. The invention also provides proteins produced by themethods of the invention. The claimed proteins include unnatural aminoacids.

The compositions and methods of the invention utilize an orthogonal tRNA(O-tRNA) aminoacyl tRNA synthetase (O-RS) pair. A wide range of pairscan be used with the following properties: the O-tRNA is preferentiallyaminoacylated with an unnatural amino acid by the O-RS. In addition, theorthogonal pair functions in the translation system of interest, e.g.,the translation system uses the unnatural amino acid-aminoacylatedO-tRNA to incorporate the unnatural amino acid into a polypeptide chain.Incorporation occurs in a site specific manner, e.g., the O-tRNArecognizes a selector codon, e.g., a stop codon, in the mRNA coding forthe protein.

In one embodiment, the O-tRNA is derived from a Tyr-tRNA from aMethanococcus jannaschii cell. In a preferred embodiment, the O-tRNA isthat referred to herein as mtRNA_(CUA) ^(Tyr). In another embodiment,the O-tRNA includes a nucleic acid polynucleotide sequence selected fromthe group that includes SEQ ID NO: 1-3 or a complementary polynucleotidesequence thereof.

In some embodiments of the invention, the O-RS is derived from TyrRSfrom a Methanococcus jannaschii cell. In a preferred embodiment, theO-RS is referred to herein as mutant TyrRS (LWJ16) or SS12-TyrRS. In afurther embodiment, the O-RS includes a polypeptide selected from thegroup consisting of a polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 35-66 and a polypeptideencoded by a nucleic acid comprising a polynucleotide sequence selectedfrom the group consisting of: SEQ ID NO: 4-34 or a complementarypolynucleotide sequence thereof.

In a preferred embodiment, the invention includes an Escherichia colicell comprising a mtRNA_(CUA) ^(Tyr) and a mutant TyrRS (LWJ16), whereinthe mutant TyrRS (LWJ16) preferentially aminoacylates the mtRNA_(CUA)^(Tyr) with O-methyl-L-tyrosine in the cell and the cell uses themtRNA_(CUA) ^(Tyr) to recognize an amber codon.

In another preferred embodiment, the invention includes an Escherichiacoli cell comprising a mtRNA_(CUA) ^(Tyr) and an SS12-TyrRS, wherein theSS12-TyrRS preferentially aminoacylates the mtRNA_(CUA) ^(Tyr) withL-3-(2-naphthyl)alanine in the cell and the cell uses the mtRNA_(CUA)^(Tyr) to recognize an amber codon.

Sequences of exemplary O-tRNA and O-RS molecules are described in theExamples.

Orthogonal tRNA and Orthogonal Aminoacyl-tRNA Synthetase Pairs

An orthogonal pair is composed of an O-tRNA, e.g., a suppressor tRNA, aframeshift tRNA, or the like, and an O-RS. The O-tRNA is not acylated byendogenous synthetases and is capable of decoding a selector codon, asdescribed above. The O-RS recognizes the O-tRNA, e.g., with an extendedanticodon loop, and preferentially aminoacylates the O-tRNA with anunnatural amino acid. The development of multiple orthogonaltRNA/synthetase pairs can allow the simultaneous incorporation ofmultiple unnatural amino acids using different codons.

The O-tRNA and the O-RS can be naturally occurring or can be derived bymutation of a naturally occurring tRNA and/or RS from a variety oforganisms, which are described under sources and hosts. In variousembodiments, the O-tRNA and O-RS are derived from at least one organism.In another embodiment, the O-tRNA is derived from a naturally occurringor mutated naturally occurring tRNA from a first organism and the O-RSis derived from naturally occurring or mutated naturally occurring RSfrom a second organism.

Methods (deriving, mutating, screening) for obtaining O-tRNA, O-RS, andpairs to be used in the compositions and methods of the invention arealso described U.S. patent application Ser. No. 10/126,931, titled“Methods and Compositions for the production of orthogonal tRNA-tRNAsynthetase pairs,” the disclosure of which is incorporated in itsentirety.

These methods solve the problems discussed in the background section forthe other strategies that were attempted to generate orthogonal tRNA/RSpairs. Specifically, these methods include: (a) generating a library oftRNAs derived from at least one tRNA from a first organism; (b)negatively selecting the library for tRNAs that are aminoacylated by anaminoacyl-tRNA synthetase (RS) from a second organism in the absence ofa RS from the first organism, thereby providing a pool of tRNAs; (c)selecting the pool of tRNAs for members that are aminoacylated by anintroduced orthogonal RS (O-RS), thereby providing at least onerecombinant O-tRNA. The at least one recombinant O-tRNA recognizes aselector codon and is not efficiency recognized by the RS from thesecond organism and is preferentially aminoacylated by the O-RS. Themethod also includes: (d) generating a library of mutant RSs derivedfrom at least one aminoacyl-tRNA synthetase (RS) from a third organism;(e) selecting the library of RSs for members that preferentiallyaminoacylate the at least one recombinant O-tRNA in the presence of anunnatural amino acid and a natural amino acid, thereby providing a poolof active RSs; and, (f) negatively selecting the pool for active RSsthat preferentially aminoacylate the at least one recombinant O-tRNA inthe absence of the unnatural amino acid, thereby providing the at leastone specific O-tRNA/O-RS pair, where the at least one specificO-tRNA/O-RS pair comprises at least one recombinant O-RS that isspecific for the unnatural amino acid and the at least one recombinantO-tRNA.

One strategy for generating an orthogonal pair involves generatingmutant libraries from which to screen and/or select an O-tRNA or O-RS.

A second strategy for generating an orthogonal tRNA/synthetase pairinvolves importing a heterologous tRNA/synthetase pair, e.g., importinga pair from another, e.g., source organism into the host cell. Theproperties of the heterologous synthetase candidate include, e.g., thatit does not charge any host cell tRNA, and the properties of theheterologous tRNA candidate include, e.g., that it is not acylated byany host cell synthetase. In addition, the heterologous tRNA derivedfrom the heterologous tRNA is orthogonal to all host cell synthetases.

Using the methods described herein and in U.S. patent application Ser.No. 10/126,931, titled “Methods and Compositions for the production oforthogonal tRNA-tRNA synthetase pairs,” the pairs and components ofpairs desired above are evolved to generate orthogonal tRNA/synthetasepairs that possess desired characteristic, e.g., that can preferentiallyaminoacylate an O-tRNA with an unnatural amino acid.

Although discussed with reference to strategies for incorporatingunnatural amino acids into proteins in vivo herein, it will beappreciated that strategies can be developed to incorporate naturalamino acids in response to selector codons as well, providing anadditional basis of and for mutagenesis. That is, a synthetase can bemodified to load a natural amino acid onto an orthogonal tRNA thatrecognizes a selector codon in a manner similar to the loading of anunnatural amino acid as described throughout.

Production of Orthogonal Aminoacyl tRNA Synthetases (O-RS)

Methods for producing an O-RS are based on generating a pool of mutantsynthetases from the framework of a wild-type synthetase, and thenselecting for mutated RSs based on their specificity for an unnaturalamino acid relative to the common twenty. To isolate such a synthetase,the selection methods of the present invention are: (i) sensitive, asthe activity of desired synthetases from the initial rounds can be lowand the population small; (ii) “tunable”, since it is desirable to varythe selection stringency at different selection rounds; and, (iii)general, so that it can be used for different unnatural amino acids.

Methods to generate an orthogonal aminoacyl tRNA synthetase includemutating the synthetase, e.g., at the active site in the synthetase, atthe editing mechanism site in the synthetase, at different sites bycombining different domains of synthetases, or the like, and applying aselection process. A strategy is used, which is based on the combinationof a positive selection followed by a negative selection. In thepositive selection, suppression of the selector codon introduced at anonessential position(s) of a positive marker allows cells to surviveunder positive selection pressure. In the presence of both natural andunnatural amino acids, survivors thus encode active synthetases chargingthe orthogonal suppressor tRNA with either a natural or unnatural aminoacid. In the negative selection, suppression of a selector codonintroduced at a nonessential position(s) of a negative marker removessynthetases with natural amino acid specificities. Survivors of thenegative and positive selection encode synthetases that aminoacylate(charge) the orthogonal suppressor tRNA with unnatural amino acids only.These synthetases can then be subjected to further mutagenesis, e.g.,DNA shuffling or other recursive mutagenesis methods.

The library of mutant RSs can be generated using various mutagenesistechniques known in the art. For example, the mutant RSs can begenerated by site-specific mutations, random point mutations, homologousrecombination, chimeric construction or the like.

The positive selection step can include, e.g., introducing a positiveselection marker, e.g., an antibiotic resistance gene, or the like, andthe library of mutant RSs into a plurality of cells, wherein thepositive selection marker comprises at least one selector codon, e.g.,an amber codon; growing the plurality of cells in the presence of aselection agent; selecting cells that survive in the presence of theselection agent by suppressing the at least one selector codon in thepositive selection marker, thereby providing a subset of positivelyselected cells that contains the pool of active mutant RSs. Optionally,the selection agent concentration can be varied.

The negative selection can include, e.g., introducing a negativeselection marker with the pool of active mutant RSs from the positiveselection into a plurality of cells of a second organism, wherein thenegative selection marker is an antibiotic resistance gene, e.g., achloramphenicol acetyltransferase (CAT) gene, comprising at least oneselector codon; and, selecting cells that survive in a 1st mediasupplemented with the unnatural amino acid and a selection agent, butfail to survive in a 2nd media not supplemented with the unnatural aminoacid and the selection agent, thereby providing surviving cells with theat least one recombinant O-RS. Optionally, the concentration of theselection agent is varied.

The positive selection can be based on suppression of a selector codonin a positive selection marker, e.g., a chloramphenicolacetyltransferase (CAT) gene comprising a selector codon, e.g., an amberstop codon, in the CAT gene, so that chloramphenicol can be applied asthe positive selection pressure. In addition, the CAT gene can be usedas both a positive marker and negative marker as describe herein in thepresence and absence of unnatural amino acid. Optionally, the CAT genecomprising a selector codon is used for the positive selection and anegative selection marker, e.g., a toxic marker, such as a barnase genecomprising at least one or more selector codons, is used for thenegative selection.

The positive selection can also be based on suppression of a selectorcodon at a nonessential position in the β-lactamase gene, renderingcells ampicillin resistant; and a negative selection using theribonuclease barnase as the negative marker is used. In contrast toβ-lactamase, which is secreted into the periplasm, CAT localizes in thecytoplasm; moreover, ampicillin is bacteriocidal, while chloramphenicolis bacteriostatic.

The recombinant O-RS can be further mutated and selected. In oneembodiment, the methods for producing at least one recombinantorthogonal aminoacyl-tRNA synthetase (O-RS) can further comprise: (d)isolating the at least one recombinant O-RS; (e) generating a second setof mutated O-RS derived from the at least one recombinant O-RS; and, (f)repeating steps (b) and (c) until a mutated O-RS is obtained thatcomprises an ability to preferentially aminoacylate the O-tRNA.Optionally, steps (d)-(f) are repeated, e.g., at least about two times.In one aspect, the second set of mutated O-RS can be generated bymutagenesis, e.g., random mutagenesis, site-specific mutagenesis,recombination or a combination thereof.

Production of Orthogonal tRNA (O-tRNAs)

Methods for producing a recombinant orthogonal tRNA (O-tRNA) areprovided in U.S. patent application Ser. No. 10/126,931, titled “Methodsand Compositions for the production of orthogonal tRNA-tRNA synthetasepairs.”

Methods of producing a recombinant O-tRNA include: (a) generating alibrary of mutant tRNAs derived from at least one tRNA, e.g., asuppressor tRNA, from a first organism; (b) negatively selecting thelibrary for mutant tRNAs that are aminoacylated by an aminoacyl-tRNAsynthetase (RS) from a second organism in the absence of a RS from thefirst organism, thereby providing a pool of mutant tRNAs; and, (c)selecting the pool of mutant tRNAs for members that are aminoacylated byan introduced orthogonal RS (O-RS), thereby providing at least onerecombinant O-tRNA; wherein the at least one recombinant O-tRNArecognizes a selector codon and is not efficiency recognized by the RSfrom the second organism and is preferentially aminoacylated by theO-RS. In one embodiment, the recombinant O-tRNA possesses an improvementof orthogonality.

For example, to improve the orthogonality of a tRNA while preserving itsaffinity toward a desired RS, the methods include a combination ofnegative and positive selections with a mutant suppressor tRNA libraryin the absence and presence of the cognate synthetase, respectively. Inthe negative selection, a selector codon(s) is introduced in a markergene, e.g., a toxic gene, such as barnase, at a nonessential position.When a member of the mutated tRNA library, e.g., derived fromMethanococcus jannaschii, is aminoacylated by endogenous host, e.g.,Escherichia coli synthetases (i.e., it is not orthogonal to the host,e.g., Escherichia coli synthetases), the selector codon, e.g., an ambercodon, is suppressed and the toxic gene product produced leads to celldeath. Cells harboring orthogonal tRNAs or non-functional tRNAs survive.Survivors are then subjected to a positive selection in which a selectorcodon, e.g., an amber codon, is placed in a positive marker gene, e.g.,a drug resistance gene, such a β-lactamase gene. These cells alsocontain an expression vector with a cognate RS. These cells are grown inthe presence of a selection agent, e.g., ampicillin. tRNAs are thenselected for their ability to be aminoacylated by the coexpressedcognate synthetase and to insert an amino acid in response to thisselector codon. Cells harboring non-functional tRNAs, or tRNAs thatcannot be recognized by the synthetase of interest are sensitive to theantibiotic. Therefore, tRNAs that: (i) are not substrates for endogenoushost, e.g., Escherichia coli, synthetases; (ii) can be aminoacylated bythe synthetase of interest; and (iii) are functional in translationsurvive both selections.

Libraries of mutated tRNA are constructed. Mutations can be introducedat a specific position(s), e.g., at a nonconservative position(s), or ata conservative position, at a randomized position(s), or a combinationof both in a desired loop of a tRNA, e.g., an anticodon loop, (D arm, Vloop, TψC arm) or a combination of loops or all loops. Chimericlibraries of tRNA are also included in the present invention. It shouldbe noted that libraries of tRNA synthetases from various organism (e.g.,microorganisms such as eubacteria or archaebacteria) such as librariesthat comprise natural diversity (see, e.g., U.S. Pat. No. 6,238,884 toShort et al; U.S. Pat. No. 5,756,316 to Schallenberger et al; U.S. Pat.No. 5,783,431 to Petersen et al; U.S. Pat. No. 5,824,485 to Thompson etal; U.S. Pat. No. 5,958,672 to Short et al), are optionally constructedand screened for orthogonal pairs.

For example, negatively selecting the library for mutant tRNAs that areaminoacylated by an aminoacyl-tRNA synthetase can include: introducing atoxic marker gene, wherein the toxic marker gene comprises at least oneof the selector codons and the library of mutant tRNAs into a pluralityof cells from the second organism; and, selecting surviving cells,wherein the surviving cells contain the pool of mutant tRNAs comprisingat least one orthogonal tRNA or nonfunctional tRNA. For example, thetoxic marker gene is a ribonuclease barnase gene, wherein theribonuclease barnase gene comprises at least one amber codon.Optionally, the ribonuclease barnase gene can include two or more ambercodons. The surviving cells can be selected, e.g., by using a comparisonratio cell density assay.

In another example, selecting the pool of mutant tRNAs for members thatare aminoacylated by an introduced orthogonal RS (O-RS) can include:introducing a positive selection marker gene, wherein the positiveselection marker gene comprises a drug resistance gene, e.g., aβ-lactamase gene, comprising at least one of the selector codons, e.g.,a β-lactamase gene comprising at least one amber stop codon, the O-RS,and the pool of mutant tRNAs into a plurality of cells from the secondorganism; and, selecting surviving cells grown in the presence of aselection agent, e.g., an antibiotic, thereby providing a pool of cellspossessing the at least one recombinant tRNA, wherein the recombinanttRNA is aminoacylated by the O-RS and inserts an amino acid into atranslation product encoded by the positive marker gene, in response tothe at least one selector codons. In another embodiment, theconcentration of the selection agent is varied. Recombinant O-tRNAsproduced by the methods are included in the present invention.

The stringency of the selection steps, e.g., the positive selectionstep, the negative selection step or both the positive and negativeselection steps, in the above described-methods, optionally includevarying the selection stringency. For example, because barnase is anextremely toxic protein, the stringency of the negative selection can becontrolled by introducing different numbers of selector codons into thebarnase gene. In one aspect of the present invention, the stringency isvaried because the desired activity can be low during early rounds.Thus, less stringent selection criteria are applied in early rounds andmore stringent criteria are applied in later rounds of selection.

Other types of selections can be used in the present invention forgenerating, e.g., O-RS, O-tRNA, and O-tRNA/O-RS pairs. For example, thepositive selection step, the negative selection step or both thepositive and negative selection steps can include using a reporter,wherein the reporter is detected by fluorescence-activated cell sorting(FACS). For example, a positive selection can be done first with apositive selection marker, e.g., chloramphenicol acetyltransferase (CAT)gene, where the CAT gene comprises a selector codon, e.g., an amber stopcodon, in the CAT gene, which followed by a negative selection screen,that is based on the inability to suppress a selector codon(s), e.g.,two or more, at positions within a negative marker, e.g., T7 RNApolymerase gene. In one embodiment, the positive selection marker andthe negative selection marker can be found on the same vector, e.g.,plasmid. Expression of the negative marker drives expression of thereporter, e.g., green fluorescent protein (GFP). The stringency of theselection and screen can be varied, e.g., the intensity of the lightneed to fluorescence the reporter can be varied. In another embodiment,a positive selection can be done with a reporter as a positive selectionmarker, which is screened by FACs, followed by a negative selectionscreen, that is based on the inability to suppress a selector codon(s),e.g., two or more, at positions within a negative marker, e.g., barnasegene.

Optionally, the reporter is displayed on a cell surface, on a phagedisplay or the like. Cell-surface display, e.g., the OmpA-basedcell-surface display system, relies on the expression of a particularepitope, e.g., a poliovirus C3 peptide fused to an outer membrane porinOmpA, on the surface of the Escherichia coli cell. The epitope isdisplayed on the cell surface only when a selector codon in the proteinmessage is suppressed during translation. The displayed peptide thencontains the amino acid recognized by one of the mutant aminoacyl-tRNAsynthetases in the library, and the cell containing the correspondingsynthetase gene can be isolated with antibodies raised against peptidescontaining specific unnatural amino acids. The OmpA-based cell-surfacedisplay system was developed and optimized by Georgiou et al. as analternative to phage display. See, Francisco, J. A., Campbell, R.,Iverson, B. L. & Georgoiu, G. Production and fluorescence-activated cellsorting of Escherichia coli expressing a functional antibody fragment onthe external surface. Proc Natl Acad Sci USA. 90:10444-8 (1993).

The selection steps can also be carried out in vitro. The selectedcomponent, e.g., synthetase and/or tRNA, can then be introduced into acell for use in in vivo incorporation of an unnatural amino acid.

Source and Host Organisms

The orthogonal tRNA-RS pair, e.g., derived from at least a first, e.g.,source organism or at least two source organisms, which can be the sameor different, can be used in a variety of host organisms, e.g., a secondorganism. The first and the second organisms of the methods of thepresent invention can be the same or different. In one embodiment, thefirst organism is a prokaryotic organism, e.g., Methanococcusjannaschii, Methanobacterium thermoautotrophicum, Halobacterium,Escherichia coli, A. fulgidus, Halobacterium, P. furiosus, P.horikoshii, A. pernix, T. thermophilus, or the like. Alternatively, thefirst organism is a eukaryotic organism, e.g., plants (e.g., complexplants such as monocots, or dicots), algae, protists, fungi (e.g.,yeast, etc), animals (e.g., mammals, insects, arthropods, etc.), or thelike. In another embodiment, the second organism is a prokaryoticorganism, Methanococcus jannaschii, Methanobacteriumthermoautotrophicum, Halobacterium, Escherichia coli, A. fulgidus,Halobacterium, P. furiosus, P. horikoshii, A. pernix, T. thermophilus,or the like. Alternatively, the second organism can be a eukaryoticorganism, e.g., plants, fungi, animals, or the like.

As described above, the individual components of a pair can be derivedfrom the same organism or different organisms. For example, tRNA can bederived from a prokaryotic organism, e.g., an archaebacterium, such asMethanococcus jannaschii and Halobacterium NRC-I or a eubacterium, suchas Escherichia coli, while the synthetase can be derived from same oranother prokaryotic organism, such as, Methanococcus jannaschii,Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, P.furiosus, P. horikoshii, A. pernix, T. thermophilus, Halobacterium,Escherichia coli or the like. Eukaryotic sources can also be used, e.g.,plants (e.g., complex plants such as monocots, or dicots), algae,protists, fungi (e.g., yeast, etc.), animals (e.g., mammals, insects,arthropods, etc.), or the like.

Selector Codons

Selector codons of the present invention expand the genetic codonframework of protein biosynthetic machinery. For example, a selectorcodon includes, e.g., a unique three base codon, a nonsense codon, suchas a stop codon, e.g., an amber codon, or an opal codon, an unnaturalcodon, at least a four base codon or the like. A number of selectorcodons can be introduced into a desired gene, e.g., one or more, two ormore, more than three, etc.

The 64 genetic codons code for 20 amino acids and 3 stop codons. Becauseonly one stop codon is needed for translational termination, the othertwo can in principle be used to encode nonproteinogenic amino acids. Theamber stop codon, UAG, has been successfully used in in vitrobiosynthetic system and in Xenopus oocytes to direct the incorporationof unnatural amino acids. Among the 3 stop codons, UAG is the least usedstop codon in Escherichia coli. Some Escherichia coli strains containnatural suppressor tRNAs, which recognize UAG and insert a natural aminoacid. In addition, these amber suppressor tRNAs have been used inconventional protein mutagenesis.

In one embodiment, the methods involve the use of a selector codon thatis a stop codon for the incorporation of unnatural amino acids in vivo.For example, an O-tRNA is generated that recognizes the stop codon,e.g., UAG, and is aminoacylated by an O-RS with a desired unnaturalamino acid. This O-tRNA is not recognized by the naturally occurringaminoacyl-tRNA synthetases. Conventional site-directed mutagenesis canbe used to introduce the stop codon, e.g., TAG, at the site of interestin the protein gene. See, e.g., Sayers, J. R., Schmidt, W. Eckstein, F.5′,3′ Exonuclease in phosphorothioate-based oligonucleotide-directedmutagenesis. Nucleic Acids Res, 791-802 (1988). When the O-RS, O-tRNAand the mutant gene are combined in vivo, the unnatural amino acid isincorporated in response to the UAG codon to give a protein containingthe unnatural amino acid at the specified position.

The incorporation of unnatural amino acids in vivo can be done withoutsignificant perturbation of the host, e.g., Escherichia coli. Forexample, because the suppression efficiency for the UAG codon dependsupon the competition between the O-tRNA, e.g., the amber suppressortRNA, and the release factor 1 (RF1) (which binds to the UAG codon andinitiates release of the growing peptide from the ribosome), thesuppression efficiency can be modulated by, e.g., either increasing theexpression level of O-tRNA, e.g., the suppressor tRNA, or using an RF1deficient strain.

Unnatural amino acids can also be encoded with rare codons. For example,when the arginine concentration in an in vitro protein synthesisreaction is reduced, the rare arginine codon, AGG, has proven to beefficient for insertion of Ala by a synthetic tRNA acylated withalanine. See, e.g., C. H. Ma, W. Kudlicki, O. W. Odom, G. Kramer and B.Hardesty, Biochemistry, 32:7939 (1993). In this case, the synthetic tRNAcompetes with the naturally occurring tRNAArg, which exists as a minorspecies in Escherichia coli. Some organisms do not use all tripletcodons. An unassigned codon AGA in Micrococcus luteus has been utilizedfor insertion of amino acids in an in vitro transcription/translationextract. See, e.g., A. K. Kowal and J. S. Oliver, Nucl. Acid. Res.,25:4685 (1997). Components of the present invention can be generated touse these rare codons in vivo.

Selector codons also comprise four or more base codons, such as, four,five, six or more base codons. Examples of four base codons include,e.g., AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codonsinclude, e.g., AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like.For example, in the presence of mutated O-tRNAs, e.g., a specialframeshift suppressor tRNAs, with anticodon loops, e.g., with at least8-10 nt anticodon loops, the four or more base codon is read as singleamino acid. In other embodiments, the anticodon loops can decode, e.g.,at least a four-base codon, at least a five-base codon, or at least asix-base codon or more. Since there are 256 possible four-base codons,multiple unnatural amino acids can be encoded in the same cell using thefour or more base codon. See, J. Christopher Anderson et al., Exploringthe Limits of Codon and Anticodon Size, Chemistry and Biology, Vol. 9,237-244 (2002); Thomas J. Magliery, Expanding the Genetic Code:Selection of Efficient Suppressors of Four-base Codons andIdentification of “Shifty” Four-base Codons with a Library Approach inEscherichia coli, J. Mol. Biol. 307: 755-769 (2001).

Methods of the present invention include using extended codons based onframeshift suppression. Four or more base codons can insert, e.g., oneor multiple unnatural amino acids into the same protein. For example,four-base codons have been used to incorporate unnatural amino acidsinto proteins using in vitro biosynthetic methods. See, e.g., C. H. Ma,W. Kudlicki, O. W. Odom, G. Kramer and B. Hardesty, Biochemistry, 1993,32, 7939 (1993); and, T. Hohsaka, D. Kajihara, Y. Ashizuka, H. Murakamiand M. Sisido, J. Am. Chem. Soc., 121:34 (1999). CGGG and AGGU were usedto simultaneously incorporate 2-naphthylalanine and an NBD derivative oflysine into streptavidin in vitro with two chemically acylatedframeshift suppressor tRNAs. See, e.g., T. Hohsaka, Y. Ashizuka, H.Sasaki, H. Murakami and M. Sisido, J. Am. Chem. Soc., 121:12194 (1999).In an in vivo study, Moore et al. examined the ability of tRNALeuderivatives with NCUA anticodons to suppress UAGN codons (N can be U, A,G, or C), and found that the quadruplet UAGA can be decoded by a tRNALeuwith a UCUA anticodon with an efficiency of 13 to 26% with littledecoding in the 0 or −1 frame. See, B. Moore, B. C. Persson, C. C.Nelson, R. F. Gesteland and J. F. Atkins, J. Mol. Biol., 298:195 (2000).In one embodiment, extended codons based on rare codons or nonsensecodons can be used in present invention, which can reduce missensereadthrough and frameshift suppression at other unwanted sites.

A translational bypassing system can also be used to incorporate anunnatural amino acid in a desired polypeptide. In a translationalbypassing system, a large sequence is inserted into a gene but is nottranslated into protein. The sequence contains a structure that servesas a cue to induce the ribosome to hop over the sequence and resumetranslation downstream of the insertion.

Alternatively, or in combination with others methods described above toincorporate an unnatural amino acid in a polypeptide, atrans-translation system can be used. This system involves a moleculecalled tmRNA present in Escherichia coli. This RNA molecule isstructurally related to an alanyl tRNA and is aminoacylated by thealanyl synthetase. The difference between tmRNA and tRNA is that theanticodon loop is replaced with a special large sequence. This sequenceallows the ribosome to resume translation on sequences that have stalledusing an open reading frame encoded within the tmRNA as template. In thepresent invention, an orthogonal tmRNA can be generated that ispreferentially aminoacylated with an orthogonal synthetase and loadedwith an unnatural amino acid. By transcribing a gene using the system,the ribosome stalls at a specific site; the unnatural amino acid isintroduced at that site, then translation resumes, using the sequenceencoded within the orthogonal tmRNA.

Selector codons optionally include unnatural base pairs. These unnaturalbase pairs further expand the existing genetic alphabet. One extra basepair increases the number of triplet codons from 64 to 125. Propertiesof third base pairs include stable and selective base pairing, efficientenzymatic incorporation into DNA with high fidelity by a polymerase, andthe efficient continued primer extension after synthesis of the nascentunnatural base pair. Descriptions of unnatural base pairs which can beadapted for methods and compositions include, e.g., Hirao, et al., Anunnatural base pair for incorporating amino acid analogues into protein,Nature Biotechnology, 20:177-182 (2002). Other relevant publications arelisted below.

For in vivo usage, the unnatural nucleoside is membrane permeable and isphosphorylated to form the corresponding triphosphate. In addition, theincreased genetic information is stable and not destroyed by cellularenzymes. Previous efforts by Benner and others took advantage ofhydrogen bonding patterns that are different from those in canonicalWatson-Crick pairs, the most noteworthy example of which is theiso-C:iso-G pair. See, e.g., C. Switzer, S. E. Moroney and S. A. Benner,J. Am. Chem. Soc., 111:8322 (1989); and, J. A. Piccirilli, T. Krauch, S.E. Moroney and S. A. Benner, Nature, 1990, 343:33 (1990); E. T. Kool,Curr. Opin. Chem. Biol., 4:602 (2000). These bases in general mispair tosome degree with natural bases and cannot be enzymatically replicated.Kool and co-workers demonstrated that hydrophobic packing interactionsbetween bases can replace hydrogen bonding to drive the formation ofbase pair. See, E. T. Kool, Curr. Opin. Chem. Biol., 4:602 (2000); and,K. M. Guckian and E. T. Kool, Angew. Chem. Int. Ed. Engl., 36, 2825(1998). In an effort to develop an unnatural base pair satisfying allthe above requirements, Schultz, Romesberg and co-workers havesystematically synthesized and studied a series of unnatural hydrophobicbases. A PICS:PICS self-pair is found to be more stable than naturalbase pairs, and can be efficiently incorporated into DNA by Klenowfragment of Escherichia coli DNA polymerase I (KF). See, e.g., D. L.McMinn, A. K. Ogawa, Y. Q. Wu, J. Q. Liu, P. G. Schultz and F. E.Romesberg, J. Am. Chem. Soc., 121:11586 (1999); and, A. K. Ogawa, Y. Q.Wu, D. L. McMinn, J. Q. Liu, P. G. Schultz and F. E. Romesberg, J. Am.Chem. Soc., 122:3274 (2000). A 3MN:3MN self-pair can be synthesized byKF with efficiency and selectivity sufficient for biological function.See, e.g., A. K. Ogawa, Y. Q. Wu, M. Berger, P. G. Schultz and F. E.Romesberg, J. Am. Chem. Soc., 122:8803 (2000). However, both bases actas a chain terminator for further replication. A mutant DNA polymerasehas been recently evolved that can be used to replicate the PICS selfpair. In addition, a 7AI self pair can be replicated. See, e.g., E. J.L. Tae, Y. Q. Wu, G. Xia, P. G. Schultz and F. E. Romesberg, J. Am.Chem. Soc., 123:7439 (2001). A novel metallobase pair, Dipic:Py, hasalso been developed, which forms a stable pair upon binding Cu(II). See,E. Meggers, P. L. Holland, W. B. Tolman, F. E. Romesberg and P. G.Schultz, J. Am. Chem. Soc., 122:10714 (2000). Because extended codonsand unnatural codons are intrinsically orthogonal to natural codons, themethods of the present invention can take advantage of this property togenerate orthogonal tRNAs for them.

Unnatural Amino Acids

As used herein an unnatural amino acid refers to any amino acid,modified amino acid, or amino acid analogue other than selenocysteineand the following twenty genetically encoded alpha-amino acids: alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine.The generic structure of an alpha-amino acid is illustrated by FormulaI:

An unnatural amino acid is typically any structure having Formula Iwherein the R group is any substituent other than one used in the twentynatural amino acids. See, e.g., Biochemistry by L. Stryer, 3^(rd) ed.1988, Freeman and Company, New York, for structures of the twentynatural amino acids. Note that, the unnatural amino acids of the presentinvention can be naturally occurring compounds other than the twentyalpha-amino acids above.

Because the unnatural amino acids of the invention typically differ fromthe natural amino acids in side chain only, the unnatural amino acidsform amide bonds with other amino acids, e.g., natural or unnatural, inthe same manner in which they are formed in naturally occurringproteins. However, the unnatural amino acids have side chain groups thatdistinguish them from the natural amino acids. For example, R in FormulaI optionally comprises an alkyl-, aryl-, acyl-, keto-, azido-,hydroxyl-, hydrazine, cyano-, halo-, hydrazide, alkenyl, alkynl, ether,thiol, seleno-, sulfonyl-, borate, boronate, phospho, phosphono,phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid,hydroxylamine, amino group, or the like or any combination thereof.Other unnatural amino acids of interest include, but are not limited to,amino acids comprising a photoactivatable cross-linker, spin-labeledamino acids, fluorescent amino acids, metal binding amino acids,metal-containing amino acids, radioactive amino acids, amino acids withnovel functional groups, amino acids that covalently or noncovalentlyinteract with other molecules, photocaged and/or photoisomerizable aminoacids, amino acids comprising biotin or a biotin analogue, glycosylatedamino acids such as a sugar substituted serine, other carbohydratemodified amino acids, keto containing amino acids, amino acidscomprising polyethylene glycol or polyether, heavy atom substitutedamino acids, chemically cleavable and/or photocleavable amino acids,amino acids with an elongated side chains as compared to natural aminoacids, e.g., polyethers or long chain hydrocarbons, e.g., greater thanabout 5 or greater than about 10 carbons, carbon-linked sugar-containingamino acids, redox-active amino acids, amino thioacid containing aminoacids, and amino acids comprising one or more toxic moiety.

In addition to unnatural amino acids that contain novel side chains,unnatural amino acids also optionally comprise modified backbonestructures, e.g., as illustrated by the structures of Formula II andIII:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′; X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids α-aminothiocarboxylates, e.g.,with side chains corresponding to the common twenty natural amino acidsor unnatural side chains. In addition, substitutions at the α-carbonoptionally include L, D, or α-α-disubstituted amino acids such asD-glutamate, D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and thelike. Other structural alternatives include cyclic amino acids, such asproline analogues as well as 3,4,6,7,8, and 9 membered ring prolineanalogues, β and γ amino acids such as substituted β-alanine and γ-aminobutyric acid.

For example, many unnatural amino acids are based on natural aminoacids, such as tyrosine, glutamine, phenylalanine, and the like.Tyrosine analogs include para-substituted tyrosines, ortho-substitutedtyrosines, and meta substituted tyrosines, wherein the substitutedtyrosine comprises an acetyl group, a benzoyl group, an amino group, ahydrazine, an hydroxyamine, a thiol group, a carboxy group, an isopropylgroup, a methyl group, a C₆-C₂₀ straight chain or branched hydrocarbon,a saturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, or the like. In addition, multiply substitutedaryl rings are also contemplated. Glutamine analogs of the inventioninclude, but are not limited to, α-hydroxy derivatives, γ-substitutedderivatives, cyclic derivatives, and amide substituted glutaminederivatives. Example phenylalanine analogs include, but are not limitedto, meta-substituted phenylalanines, wherein the substituent comprises ahydroxy group, a methoxy group, a methyl group, an allyl group, anacetyl group, or the like. Specific examples of unnatural amino acidsinclude, but are not limited to, O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, and anisopropyl-L-phenylalanine, and the like. The structures of a variety ofunnatural amino acids are provided in the figures, e.g., FIGS. 17, 18,19, 26, and 29.

Typically, the unnatural amino acids of the invention are selected ordesigned to provide additional characteristics unavailable in the twentynatural amino acids. For example, unnatural amino acid are optionallydesigned or selected to modify the biological properties of a protein,e.g., into which they are incorporated. For example, the followingproperties are optionally modified by inclusion of an unnatural aminoacid into a protein: toxicity, biodistribution, solubility, stability,e.g., thermal, hydrolytic, oxidative, resistance to enzymaticdegradation, and the like, facility of purification and processing,structural properties, spectroscopic properties, chemical and/orphotochemical properties, catalytic activity, redox potential,half-life, ability to react with other molecules, e.g., covalently ornoncovalently, and the like.

Chemical Synthesis of Unnatural Amino Acids

Many of the unnatural amino acids provided above are commerciallyavailable, e.g., from Sigma (USA) or Aldrich (Milwaukee, Wis., USA).Those that are not commercially available are optionally synthesized asprovided in the examples below or using standard methods known to thoseof skill in the art. For organic synthesis techniques, see, e.g.,Organic Chemistry by Fessendon and Fessendon, (1982, Second Edition,Willard Grant Press, Boston Mass.); Advanced Organic Chemistry by March(Third Edition, 1985, Wiley and Sons, New York); and Advanced OrganicChemistry by Carey and Sundberg (Third Edition, Parts A and B, 1990,Plenum Press, New York).

For example, meta-substituted phenylalanines are synthesized in aprocedure as outlined in FIG. 14. Typically, NBS (N-bromosuccinimide) isadded to a meta-substituted methylbenzene compound to give ameta-substituted benzyl bromide, which is then reacted with a malonatecompound to give the meta substituted phenylalanine. Typicalsubstituents used for the meta position include, but are not limited to,ketones, methoxy groups, alkyls, acetyls, and the like. For example,3-acetyl-phenylalanine is made by reacting NBS with a solution of3-methylacetophenone. For more details see the examples below. A similarsynthesis is used to produce a 3-methoxy phenylalanine. The R group onthe meta position of the benzyl bromide in that case is —OCH₃. See,e.g., Matsoukas et al., J. Med. Chem., 1995, 38, 4660-4669.

In some embodiments, the design of unnatural amino acids is biased byknown information about the active sites of synthetases, e.g.,orthogonal tRNA synthetases used to aminoacylate an orthogonal tRNA. Forexample, three classes of glutamine analogs are provided, includingderivatives substituted at the nitrogen of amide (1), a methyl group atthe γ-position (2), and a N-C^(γ)-cyclic derivative (3). Based upon thex-ray crystal structure of E. coli GlnRS, in which the key binding siteresidues are homologous to yeast GlnRS, the analogs were designed tocomplement an array of side chain mutations of residues within a 10 Åshell of the side chain of glutamine, e.g., a mutation of the activesite Phe233 to a small hydrophobic amino acid might be complemented byincreased steric bulk at the C^(γ) position of Gln.

For example, N-phthaloyl-L-glutamic 1,5-anhydride (compound number 4 inFIG. 23) is optionally used to synthesize glutamine analogs withsubstituents at the nitrogen of the amide. See, e.g., King, F. E. &Kidd, D. A. A. A New Synthesis of Glutamine and of γ-Dipeptides ofGlutamic Acid from Phthylated Intermediates. J. Chem. Soc., 3315-3319(1949); Friedman, O. M. & Chatterrji, R. Synthesis of Derivatives ofGlutamine as Model Substrates for Anti-Tumor Agents. J. Am. Chem. Soc.81, 3750-3752 (1959); Craig, J. C. et al. Absolute Configuration of theEnantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl]amino]quinoline (Chloroquine). J. Org.Chem. 53, 1167-1170 (1988); and Azoulay, M., Vilmont, M. & Frappier, F.Glutamine analogues as Potential Antimalarials, Eur. J. Med. Chem. 26,201-5 (1991). The anhydride is typically prepared from glutamic acid byfirst protection of the amine as the phthalimide followed by refluxingin acetic acid. The anhydride is then opened with a number of amines,resulting in a range of substituents at the amide. Deprotection of thephthaloyl group with hydrazine affords a free amino acid as shown inFIG. 23.

Substitution at the γ-position is typically accomplished via alkylationof glutamic acid. See, e.g., Koskinen, A. M. P. & Rapoport, H. Synthesisof 4-Substituted Prolines as Conformationally Constrained Amino AcidAnalogues. J. Org. Chem. 54, 1859-1866. (1989). A protected amino acid,e.g., as illustrated by compound number 5 in FIG. 24 is optionallyprepared by first alkylation of the amino moiety with9-bromo-9-phenylfluorene (PhflBr) (see, e.g., Christie, B. D. &Rapoport, H. Synthesis of Optically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)-Apovincamine through AminoAcid Decarbonylation and Iminium Ion Cyclization. J. Org. Chem. 1989,1859-1866 (1985)) and then esterification of the acid moiety usingO-tert-butyl-N,N′-diisopropylisourea. Addition of KN(Si(CH₃)₃)₂regioselectively deprotonates at the α-position of the methyl ester toform the enolate, which is then optionally alkylated with a range ofalkyl iodides. Hydrolysis of the t-butyl ester and Phfl group gave thedesired γ-methyl glutamine analog (Compound number 2 in FIG. 24).

An N—C^(γ) cyclic analog, as illustrated by Compound number 3 in FIG.25, is optionally prepared in 4 steps from Boc-Asp-Ot-Bu as previouslydescribed. See, e.g., Barton, D. H. R., Herve, Y., Potier, P. & Thierry,J. Synthesis of Novel a-Amino-Acids and Derivatives Using RadicalChemistry: Synthesis of L- and D-a-Amino-Adipic Acids, L-a-aminopimelicAcid and Appropriate Unsaturated Derivatives. Tetrahedron Lett. 43,4297-4308 (1987) and, Subasinghe, N., Schulte, M., Roon, R. J., Koerner,J. F. & Johnson, R. L. Quisqualic acid analogues: synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activityat a novel quisqualate-sensitized site. J. Med. Chem. 35 4602-7 (1992).Generation of the anion of the N-t-Boc-pyrrolidinone, pyrrolidinone, oroxazolidone followed by the addition of the compound 7, as shown in FIG.25, results in a Michael addition product. Deprotection with TFA thenresults in the free amino acids.

In addition to the above unnatural amino acids, a library of tyrosineanalogs has also been designed. Based upon the crystal structure of B.stearothermophilus TyrRS, whose active site is highly homologous to thatof the M. jannashii synthetase, residues within a 10 Å shell of thearomatic side chain of tyrosine were mutated (Y32, G34, L65, Q155, D158,A167, Y32 and D158). The library of tyrosine analogs, as shown in FIG.26, has been designed to complement an array of substitutions to theseactive site amino acids. These include a variety of phenyl substitutionpatterns, which offer different hydrophobic and hydrogen-bondingproperties. Tyrosine analogs are optionally prepared using the generalstrategy illustrated by FIG. 27. For example, an enolate of diethylacetamidomalonate is optionally generated using sodium ethoxide. Adesired tyrosine analog can then be prepared by adding an appropriatebenzyl bromide followed by hydrolysis.

Cellular Uptake of Unnatural Amino Acids

Unnatural amino acid uptake is one issue that is typically consideredwhen designing and selecting unnatural amino acids, e.g., forincorporation into a protein. For example, the high charge density ofα-amino acids suggests that these compounds are unlikely to be cellpermeable. Natural amino acids are taken up into bacteria via acollection of protein-based transport systems displaying varying degreesof amino acid specificity. The present invention therefore provides arapid screen for assessing which unnatural amino acids, if any, aretaken up by cells.

For example, a variety of unnatural amino acids are optionally screenedin minimal media for toxicity to cells. Toxicities are typically sortedinto five groups: (1) no toxicity, in which no significant change indoubling times occurs; (2) low toxicity, in which doubling timesincrease by less than about 10%; (3) moderate toxicity, in whichdoubling times increase by about 10% to about 50%; (4) high toxicity, inwhich doubling times increase by about 50% to about 100%; and (5)extreme toxicity, in which doubling times increase by more than about100%. See, e.g., Liu, D. R. & Schultz, P. G. Progress toward theevolution of an organism with an expanded genetic code. Proceedings ofthe National Academy of Sciences of the United States of America 96,4780-4785 (1999). The toxicity of the amino acids scoring as highly orextremely toxic is typically measured as a function of theirconcentration to obtain IC50 values. In general, amino acids which arevery close analogs of natural amino acids or which display reactivefunctionality demonstrate the highest toxicities. The former trendsuggests that mechanisms of toxicity for these unnatural amino acids canbe incorporation into proteins or inhibition of essential enzymes thatprocess natural amino acids.

To identify possible uptake pathways for toxic amino acids, toxicityassays are optionally repeated at IC50 levels, e.g., in mediasupplemented with an excess of a structurally similar natural aminoacid. For toxic amino acids, the presence of excess natural amino acidtypically rescues the ability of the cells to grow in the presence ofthe toxin, presumably because the natural amino acid effectivelyoutcompetes the toxin for either cellular uptake or for binding toessential enzymes. In these cases, the toxic amino acid is optionallyassigned a possible uptake pathway and labeled a “lethal allele” whosecomplementation is required for cell survival. These lethal alleles areextremely useful for assaying the ability of cells to uptake nontoxicunnatural amino acids. Complementation of the toxic allele, evidenced bythe restoration of cell growth, suggests that the nontoxic amino acid istaken up by the cell, possibly by the same uptake pathway as thatassigned to the lethal allele. A lack of complementation isinconclusive. For example studies and conclusions see the examplesprovided below.

Results obtained, e.g., as described in the examples below, demonstratethat complementation of lethal unnatural amino acid alleles is anefficient method for qualitatively assessing amino acid uptake. Themethod typically requires far less effort than radiolabeling largenumbers of compounds and is therefore a more advantageous method foranalyzing unnatural; amino acids of interest. This general strategy isoptionally used to rapidly evaluate the cellular uptake of a wide rangeof molecules such as nucleic acid base analogs, carbohydrate analogs, orpeptide analogs. For example, this strategy is optionally used toevaluate the cellular uptake of the unnatural amino aids presentedherein.

The present invention also provides a general method for deliveringunnatural amino acids, which is independent of all amino acid uptakepathways. This general method relies on uptake via peptide permeases,which transport dipeptides and tripeptides across the cytoplasmicmembrane. Peptide permeases are not very side-chain specific, and the KDvalues for their substrates are comparable to KD values of amino acidpermeases, e.g., about 0.1 mM to about 10 mM). See, e.g., Nickitenko,A., Trakhanov, S. & Quiocho, S. A structure of DppA, a periplasmicdepeptide transport/chemosensory receptor. Biochemistry 34, 16585-16595(1995) and Dunten, P., Mowbray, S. L. Crystal structure of the dipeptidebinding protein from Escherichia coli involved in active transport andchemotaxis. Protein Science 4, 2327-34 (1995). The unnatural amino acidsare then uptaken as conjugates of natural amino acids, such as lysine,and released into the cytoplasm upon hydrolysis of the dipeptide by oneof endogenous E. coli peptidases. To test this approach, we synthesizedseveral Unn-Lys and Lys-Unn dipeptides by solid phase synthesis, andtested the growth of an E. coli strain deficient in lysine biosynthesison lysine minimal media in the presence and absence of these dipeptides.The only source of lysine available to these cells is the dipeptidecontaining the unnatural amino acid. Uptake of phosphonoserine,phosphonotyrosine, pentafluorophenylalanine, and caged serine have beenanalyzed in this manner. In all four cases, growth was observed on 10 mMand higher dipeptide concentrations. Although uptake is easily analyzedwith the method provided herein, an alternative to designing unnaturalamino acid that are amenable to cellular uptake pathways, is to providebiosynthetic pathways to create amino acids in vivo.

Biosynthesis of Unnatural Amino Acids

Many biosynthetic pathways already exist in cells for the production ofamino acids and other compounds. While a biosynthetic method for aparticular unnatural amino acid may not exist in nature, e.g., in E.coli, the present invention provide such methods. For example,biosynthetic pathways for unnatural amino acids are optionally generatedin E. coli by adding new enzymes or modifying existing E. coli pathways.Additional new enzymes are optionally naturally occurring enzymes orartificially evolved enzymes. For example, the biosynthesis ofp-aminophenylalanine (as presented in an example below) relies on theaddition of a combination of known enzymes from other organisms. Thegenes for these enzymes can be introduced into a cell, e.g., an E. colicell, by transforming the cell with a plasmid comprising the genes. Thegenes, when expressed in the cell, provide an enzymatic pathway tosynthesize the desired compound. Examples of the types of enzymes thatare optionally added are provided in the examples below. Additionalenzymes sequences are found, e.g., in Genbank. Artificially evolvedenzymes are also optionally added into a cell in the same manner. Inthis manner, the cellular machinery and resources of a cell aremanipulated to produce unnatural amino acids.

A variety of methods are available for producing novel enzymes for usein biosynthetic pathways or for evolution of existing pathways. Forexample, recursive recombination, e.g., as developed by Maxygen, Inc.(on the world wide web at maxygen.com), is optionally used to developnovel enzymes and pathways. See, e.g., Stemmer 1994, “Rapid evolution ofa protein in vitro by DNA shuffling,” Nature Vol. 370 No. 4: Pg.389-391; and Stemmer, 1994, “DNA shuffling by random fragmentation andreassembly: In vitro recombination for molecular evolution,” Proc. Natl.Acad. Sci. USA. Vol. 91: Pg. 10747-10751. Similarly DesignPath™,developed by Genencor (on the world wide web at genencor.com) isoptionally used for metabolic pathway engineering, e.g., to engineer apathway to create O-methyl-L-trosine in E coli. This technologyreconstructs existing pathways in host organisms using a combination ofnew genes, e.g., identified through functional genomics, and molecularevolution and design. Diversa Corporation (on the world wide web atdiversa.com) also provides technology for rapidly screening libraries ofgenes and gene pathways, e.g., to create new pathways.

Typically, the biosynthesis methods of the present invention, e.g., thepathway to create p-aminophenylalanine (pAF) from chorismate, do notaffect the concentration of other amino acids produced in the cell. Forexample a pathway used to produce pAF from chorismate produces pAF inthe cell while the concentrations of other aromatic amino acidstypically produced from chorismate are not substantially affected.Typically the unnatural amino acid produced with an engineeredbiosynthetic pathway of the present invention is produced in aconcentration sufficient for efficient protein biosynthesis, e.g., anatural cellular amount, but not to such a degree as to affect theconcentration of the other amino acids or exhaust cellular resources.Typical concentrations produced in vivo in this manner are about 10 mMto about 0.05 mM. Once a bacterium is transformed with a plasmidcomprising the genes used to produce enzymes desired for a specificpathway and a twenty-first amino acid, e.g., pAF, dopa,O-methyl-L-tyrosine, or the like, is generated, in vivo selections areoptionally used to further optimize the production of the unnaturalamino acid for both ribosomal protein synthesis and cell growth.

Compositions that Include Proteins with Unnatural Amino Acids

The invention provides compositions of matter, including proteins withat least one unnatural amino acid. The invention also providescompositions of matter that include proteins with at least one unnaturalamino acid produced using the compositions and methods of the invention.In one embodiment, the proteins are processed and modified in a celldependent manner, e.g., phosphorylated, glycosylated, folded, membranebound, etc.

In one aspect, the composition optionally includes at least about 10micrograms, e.g., at least about 50 micrograms, at least about 100micrograms, at least about 500 micrograms, at least about 1 milligram,or even at least about 10 milligrams or more of the protein, e.g., anamount that can be achieved with in vivo protein production methods(details on recombinant protein production and purification are providedherein). For example, the protein is optionally present in thecomposition at a concentration of at least about 10 micrograms perliter, at least about 50 micrograms per liter, at least about 100micrograms per liter, at least about 500 micrograms per liter, at leastabout 1 milligram per liter, or at least about 10 milligrams per literof the protein, or more micrograms or protein per liter, e.g., in a celllysate, pharmaceutical buffer, or other liquid suspension (e.g., in avolume of, e.g., anywhere from about 1 nl to about 100 L). Theproduction of large quantities (e.g., greater that that typicallypossible with other methods, e.g., in vitro translation) of a proteinincluding at least one unnatural amino acid is a feature of theinvention and is an advantage over the prior art.

The production of large quantities (e.g., greater that that typicallypossible with other methods, e.g., in vitro translation) of a proteinincluding at least one unnatural amino acid is a feature of theinvention and is an advantage over the prior art. For example, theability to synthesize large quantities of proteins containing, e.g.,heavy atoms, facilitates protein structure determination via, e.g.,X-ray crystallography.

The incorporation of an unnatural amino acid can be done to, e.g.,tailor changes in protein structure and/or function, e.g., to changesize, acidity, nucleophilicity, hydrogen bonding, hydrophobicity,accessibility of protease target sites, etc. Proteins that include anunnatural amino acid can have enhanced or even entirely new catalytic orphysical properties. For example, the following properties areoptionally modified by inclusion of an unnatural amino acid into aprotein: toxicity, biodistribution, structural properties, spectroscopicproperties, chemical and/or photochemical properties, catalytic ability,half-life (e.g., serum half-life), ability to react with othermolecules, e.g., covalently or noncovalently, and the like. Thecompositions including proteins that include at least one unnaturalamino acid are useful for, e.g., novel therapeutics, diagnostics,catalytic enzymes, binding proteins (e.g., antibodies), and e.g., thestudy of protein structure and function.

In one aspect of the invention, a composition includes at least oneprotein with at least one, e.g., at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least ten, or more unnatural amino acids. For a givenprotein with more than one unnatural amino acids, the unnatural aminoacids can be identical or different (e.g., the protein can include twoor more different types of unnatural amino acids, or can include two ormore different sites having unnatural amino acids, or both).

Essentially any protein that includes an unnatural amino acid (and anycorresponding coding nucleic acid, e.g., which includes one or moreselector codons) can be produced using the compositions and methodsherein. No attempt is made to identify the hundreds of thousands ofknown proteins, any of which can be modified to include one or moreunnatural amino acid, e.g., by tailoring any available mutation methodsto include one or more appropriate selector codon in a relevanttranslation system. Common sequence repositories for known proteinsinclude GenBank EMBL, DDBJ and the NCBI. Other repositories can easilybe identified by searching the internet.

One preferred class of proteins that can be made using the compositionsand methods for in vivo incorporation of unnatural amino acids describedherein includes therapeutic proteins. Examples of therapeutic and otherproteins that can be modified to comprise one or more unnatural include,e.g., Alpha-1 antitrypsin, Angiostatin, Antihemolytic factor, antibodies(further details on antibodies are found below), Apolipoprotein,Apoprotein, Atrial natriuretic factor, Atrial natriuretic polypeptide,Atrial peptides, C—X—C chemokines (e.g., T39765, NAP-2, ENA-78, Gro-a,Gro-b, Gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG), Calcitonin, CCchemokines (e.g., Monocyte chemoattractant protein-1, Monocytechemoattractant protein-2, Monocyte chemoattractant protein-3, Monocyteinflammatory protein-1 alpha, Monocyte inflammatory protein-1 beta,RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262), CD40ligand, C-kit Ligand, Collagen, Colony stimulating factor (CSF),Complement factor 5a, Complement inhibitor, Complement receptor 1,cytokines, (e.g., epithelial Neutrophil Activating Peptide-78,GROα/MGSA, GROβ, GROγ, MIP-1α, MIP-16, MCP-1), Epidermal Growth Factor(EGF), Erythropoietin (“EPO”, representing a preferred target formodification by the incorporation of one or more unnatural amino acid),Exfoliating toxins A and B, Factor IX, Factor VII, Factor VIII, FactorX, Fibroblast Growth Factor (FGF), Fibrinogen, Fibronectin, G-CSF,GM-CSF, Glucocerebrosidase, Gonadotropin, growth factors, Hedgehogproteins (e.g., Sonic, Indian, Desert), Hemoglobin, Hepatocyte GrowthFactor (HGF), Hirudin, Human serum albumin, Insulin, Insulin-like GrowthFactor (IGF), interferons (e.g., IFN-α, IFN-β, IFN-γ), interleukins(e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, etc.), Keratinocyte Growth Factor (KGF), Lactoferrin,leukemia inhibitory factor, Luciferase, Neurturin, Neutrophil inhibitoryfactor (NIF), oncostatin M, Osteogenic protein, Parathyroid hormone,PD-ECSF, PDGF, peptide hormones (e.g., Human Growth Hormone),Pleiotropin, Protein A, Protein G, Pyrogenic exotoxins A, B, and C,Relaxin, Renin, SCF, Soluble complement receptor I, Soluble I-CAM 1,Soluble interleukin receptors (IL-1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12,13, 14, 15), Soluble TNF receptor, Somatomedin, Somatostatin,Somatotropin, Streptokinase, Superantigens, i.e., Staphylococcalenterotoxins (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE), Superoxidedismutase, Toxic shock syndrome toxin (TSST-1), Thymosin alpha 1, Tissueplasminogen activator, Tumor necrosis factor beta (TNF beta), Tumornecrosis factor receptor (TNFR), Tumor necrosis factor-alpha (TNFalpha), Vascular Endothelial Growth Factor (VEGEF), Urokinase and manyothers.

Many of these proteins are commercially available (See, e.g., the SigmaBioSciences 2002 catalogue and price list), and the correspondingprotein sequences and genes and, typically, many variants thereof, arewell-known (see, e.g., Genebank). Any of them can be modified by theinsertion of one or more unnatural amino acid according to the presentinvention, e.g., to alter the protein with respect to one or moretherapeutic property of interest. Examples of therapeutically relevantproperties include serum half-life, shelf half-life, stability,immunogenicity, therapeutic activity, detectability (e.g., by theinclusion of reporter groups (e.g., labels or label binding sites) inthe unnatural amino acids), reduction of LD-50 or other side effects,ability to enter the body through the gastric tract (e.g., oralavailability), or the like.

One class of proteins that can be made using the compositions andmethods for in vivo incorporation of unnatural amino acids describedherein includes transcriptional and expression activators. Exampletranscriptional and expression activators include genes and proteinsthat modulate cell growth, differentiation, regulation, or the like.Expression and transcriptional activators are found in prokaryotes,viruses, and eukaryotes, including fungi, plants, and animals, includingmammals, providing a wide range of therapeutic targets. It will beappreciated that expression and transcriptional activators regulatetranscription by many mechanisms, e.g., by binding to receptors,stimulating a signal transduction cascade, regulating expression oftranscription factors, binding to promoters and enhancers, binding toproteins that bind to promoters and enhancers, unwinding DNA, splicingpre-mRNA, polyadenylating RNA, and degrading RNA.

One preferred class of proteins of the invention (e.g., proteins withone or more unnatural amino acids) include expression activators such ascytokines, inflammatory molecules, growth factors, their receptors, andoncogene products, e.g., interleukins (e.g., IL-1, IL-2, IL-8, etc.),interferons, FGF, IGF-I, IGF-II, FGF, PDGF, TNF, TGF-α, TGF-β, EGF, KGF,SCF/c-Kit, CD40UCD40, VLA-4/VCAM-1, ICAM-1/LFA-1, and hyalurin/CD44;signal transduction molecules and corresponding oncogene products, e.g.,Mos, Ras, Raf, and Met; and transcriptional activators and suppressors,e.g., p53, Tat, Fos, Myc, Jun, Myb, Rel, and steroid hormone receptorssuch as those for estrogen, progesterone, testosterone, aldosterone, theLDL receptor ligand and corticosterone.

A variety of other proteins can also be modified to include one or moreunnatural amino acid of the invention. For example, the invention caninclude substituting one or more natural amino acids in one or morevaccine proteins with an unnatural amino acid, e.g., in proteins frominfectious fungi, e.g., Aspergillus, Candida species; bacteria,particularly E. coli, which serves a model for pathogenic bacteria, aswell as medically important bacteria such as Staphylococci (e.g.,aureus), or Streptococci (e.g., pneumoniae); protozoa such as sporozoa(e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates(Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viruses such as(+) RNA viruses (examples include Poxviruses e.g., vaccinia;Picornaviruses, e.g. polio; Togaviruses, e.g., rubella; Flaviviruses,e.g., HCV; and Coronaviruses), (−) RNA viruses (e.g., Rhabdoviruses,e.g., VSV; Paramyxovimses, e.g., RSV; Orthomyxovimses, e.g., influenza;Bunyaviruses; and Arenaviruses), dsDNA viruses (Reoviruses, forexample), RNA to DNA viruses, i.e., Retroviruses, e.g., HIV and HTLV,and certain DNA to RNA viruses such as Hepatitis B.

A variety of enzymes (e.g., industrial enzymes) can also be modified toinclude one or more unnatural amino acid according to the methodsherein, such as amidases, amino acid racemases, acylases, dehalogenases,dioxygenases, diarylpropane peroxidases, epimerases, epoxide hydrolases,esterases, isomerases, kinases, glucose isomerases, glycosidases,glycosyl transferases, haloperoxidases, monooxygenases (e.g., p450s),lipases, lignin peroxidases, nitrile hydratases, nitrilases, proteases,phosphatases, subtilisins, transaminase, and nucleases.

Agriculturally related proteins such as insect resistance proteins(e.g., the Cry proteins), starch and lipid production enzymes, plant andinsect toxins, toxin-resistance proteins, Mycotoxin detoxificationproteins, plant growth enzymes (e.g., Ribulose 1,5-BisphosphateCarboxylase/Oxygenase, “RUBISCO”), lipoxygenase (LOX), andPhosphoenolpyruvate (PEP) carboxylase are also suitable targets forunnatural amino acid modification.

Genes coding for proteins including at least one unnatural amino acidcan be mutagenized using methods well-known to one of skill in the artand described herein under “General Molecular Biology Techniques.” Forexample, a nucleic acid for a protein of interest is mutagenized toinclude one or more selector codon, providing for insertion of the oneor more unnatural amino acids. The present invention includes any suchvariant, e.g., mutant, versions of any protein, e.g., including at leastone unnatural amino acid.

Similarly, the present invention also includes corresponding nucleicacids, i.e., any nucleic acid with one or more selector codon thatencodes one or more unnatural amino acid.

In one example embodiment, the invention provides compositions thatinclude a Asp112TAG mutant of chloramphenicol acetyltransferase (CAT)produced by the compositions and methods of the invention, where the CATprotein includes at least one unnatural amino acid, e.g., anO-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, an amino-, isopropyl-,or allyl-containing tyrosine analogue, etc., and the protein is presentin the composition at a concentration of at least about 100 microgramsper liter. In another embodiment, the invention provides compositionsthat include a Tyr163TAG mutant of mouse dihydrofolate reductase (DHFR)where the DHFR protein includes at least one unnatural amino, e.g., anO-methyl-L-tyrosine, an L-3-(2-naphthyl)alanine, an amino-, isopropyl-,or allyl-containing tyrosine analogue, etc., and the protein is presentin the composition at a concentration of at least about 100 microgramsper liter.

Making Antibodies to Proteins Comprising Unnatural Amino Acids

In one aspect, the present invention provides antibodies to unnaturalamino acids and to proteins comprising unnatural amino acids. Antibodiesto unnatural amino acids and proteins comprising such unnatural aminoacids are useful as purification reagents, e.g., for purifying theproteins and unnatural amino acids of the invention. In addition, theantibodies can be used as indicator reagents to indicate the presence ofan unnatural amino acid or protein comprising an unnatural amino acid,e.g., to track the presence or location (e.g., in vivo or in situ) ofthe unnatural amino acid or protein comprising an unnatural amino acid.It is also, of course, the case that the unnatural amino acid can itselfcomprise one or more unnatural amino acids, thereby providing anantibody with one or more property conferred by the one or moreunnatural amino acids.

An antibody of the invention can be a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (e.g., antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (V_(H)) refer to these light and heavy chains,respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab′)₂, a dimer of Fab whichitself is a light chain joined to V_(H)-C_(H)1 by a disulfide bond. TheF(ab′)₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the F(ab′)₂ dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1999), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments, etc. may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein, also optionally includes antibody fragments either produced bythe modification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Antibodies include single chainantibodies, including single chain Fv (sFv or scFv) antibodies in whicha variable heavy and a variable light chain are joined together(directly or through a peptide linker) to form a continuous polypeptide.Antibodies of the invention can be, e.g., polyclonal, monoclonal,chimeric, humanized, single chain, Fab fragments, fragments produced byan Fab expression library, or the like.

In general, antibodies of the invention are valuable, both as generalreagents and as therapeutic reagents in a variety of molecularbiological or pharmaceutical processes. Methods of producing polyclonaland monoclonal antibodies are available, and can be applied to makingthe antibodies of the present invention. A number of basic textsdescribe standard antibody production processes, including, e.g.,Borrebaeck (ed) (1995) Antibody Engineering, 2^(nd) Edition Freeman andCompany, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering,A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty),and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, NJ(Paul); Paul (ed.), (1999) Fundamental Immunology, Fifth edition RavenPress, N.Y.; Coligan (1991) Current Protocols in ImmunologyWiley/Greene, NY; Harlow and Lane (1989) Antibodies: A Laboratory ManualCold Spring Harbor Press, NY; Stites et al. (eds.) Basic and ClinicalImmunology (4th ed.) Lange Medical Publications, Los Altos, Calif., andreferences cited therein; Goding (1986) Monoclonal Antibodies:Principles and Practice (2d ed.) Academic Press, New York, N.Y.; andKohler and Milstein (1975) Nature 256: 495-497.

A variety of recombinant techniques for antibody preparation which donot rely on, e.g., injection of an antigen into an animal have beendeveloped and can be used in the context of the present invention. Forexample, it is possible to generate and select libraries of recombinantantibodies in phage or similar vectors. See, e.g., Winter et al. (1994)“Making Antibodies by Phage Display Technology” Annu. Rev. Immunol.12:433-55 and the references cited therein for a review. See also,Griffiths and Duncan (1998) “Strategies for selection of antibodies byphage display” Curr Opin Biotechnol 9: 102-8; Hoogenboom et al. (1998)“Antibody phage display technology and its applications”Immunotechnology 4: 1-20; Gram et al. (1992) “in vitro selection andaffinity maturation of antibodies from a naïve combinatorialimmunoglobulin library” PNAS 89:3576-3580; Huse et al. (1989) Science246: 1275-1281; and Ward, et al. (1989) Nature 341: 544-546.

In one embodiment, antibody libraries can include repertoires of V genes(e.g., harvested from populations of lymphocytes or assembled in vitro)which are cloned for display of associated heavy and light chainvariable domains on the surface of filamentous bacteriophage. Phage areselected by binding to an antigen. Soluble antibodies are expressed fromphage infected bacteria and the antibody can be improved, e.g., viamutagenesis. See e.g., Balint and Larrick (1993) “Antibody Engineeringby Parsimonious Mutagenesis” Gene 137:109-118; Stemmer et al. (1993)“Selection of an Active Single Chain Fv Antibody From a Protein LinkerLibrary Prepared by Enzymatic Inverse PCR” Biotechniques 14(2):256-65;Crameri et al. (1996) “Construction and evolution of antibody-phagelibraries by DNA shuffling” Nature Medicine 2:100-103; and Crameri andStemmer (1995) “Combinatorial multiple cassette mutagenesis creates allthe permutations of mutant and wildtype cassettes” BioTechniques18:194-195.

Kits for cloning and expression of recombinant antibody phage systemsare also known and available, e.g., the “recombinant phage antibodysystem, mouse ScFv module,” from Amersham-Pharmacia Biotechnology(Uppsala, Sweden). Bacteriophage antibody libraries have also beenproduced for making high affinity human antibodies by chain shuffling(See, e.g., Marks et al. (1992) “By-Passing Immunization: Building HighAffinity Human Antibodies by Chain Shuffling” Biotechniques 10:779-782.Indeed, antibodies can typically be custom ordered from any of a varietyof sources, such as PeptidoGenic (pkim@ccnet on the commercialdomain(.com), HTI Bio-products, inc. (on the world-wide web (www) athtibio on the commercial domain(.com)), BMA Biomedicals Ltd (U.K.), Bio.Synthesis, Inc., Research Genetics (Huntsville, Ala.) and many others.

In certain embodiments, it is useful to “humanize” antibodies of theinvention, e.g., where the antibodies are to be administeredtherapeutically. The use of humanized antibodies tends to reduce theincidence of unwanted immune responses against the therapeuticantibodies (e.g., when the patient is a human). The antibody referencesabove describe humanization strategies. In addition to humanizedantibodies, human antibodies are also a feature of the invention. Humanantibodies consist of characteristically human immunoglobulin sequences.Human antibodies can be produced in using a wide variety of methods(see, e.g., Larrick et al., U.S. Pat. No. 5,001,065, for a review). Ageneral approach for producing human antibodies by trioma technology isdescribed by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S.Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666.

A variety of methods of using antibodies in the purification anddetection of proteins are known and can be applied to detecting andpurifying proteins comprising unnatural amino acids as noted herein. Ingeneral, antibodies are useful reagents for ELISA, western blotting,immunochemistry, affinity chromatography methods, SPR, and many othermethods. The references noted above provide details on how to performELISA assays, western blots, surface plasmon resonance (SPR) and thelike.

In one aspect of the invention, antibodies of the invention themselvesinclude unnatural amino acids, providing the antibodies with propertiesof interest (e.g., improved half-life, stability, toxicity, or the like.Antibodies account for nearly 50% of all compounds currently in clinicaltrials (Wittrup, (1999) “Phage on display” Tibtech 17: 423-424 andantibodies are used ubiquitously as diagnostic reagents. Accordingly,the ability to modify antibodies with unnatural amino acids provides animportant tool for modifying these valuable reagents.

For example, there are many applications of MAbs to the field ofdiagnostics. Assays range from simple spot tests to more involvedmethods such as the radio-labeled NR-LU-10 MAb from DuPont Merck Co.used for tumor imaging (Rusch et al. (1993) “NR-LU-10 monoclonalantibody scanning. A helpful new adjunct to computed tomography inevaluating non-small-cell lung cancer.” J Thorac Cardiovasc Surg 106:200-4). As noted, MAbs are central reagents for ELISA, western blotting,immunochemistry, affinity chromatography methods and the like. Any suchdiagnostic antibody can be modified to include one or more unnaturalamino acid, altering, e.g., the specificity or avidity of the Ab for atarget, or altering one or more detectable property, e.g., by includinga detectable label (e.g., spectrographic, fluorescent, luminescent,etc.) in the unnatural amino acid.

One class of valuable antibody reagents are therapeutic Abs. Forexample, antibodies can be tumor-specific MAbs that arrest tumor growthby Targeting tumor cells for destruction by antibody-dependentcell-mediated cytotoxicity (ADCC) or complement-mediated lysis (CML)(these general types of Abs are sometimes referred to as “magicbullets”). One example is Rituxan, an anti-CD20 MAb for the treatment ofNon-Hodgkins lymphoma (Scott (1998) “Rituximab: a new therapeuticmonoclonal antibody for non-Hodgkin's lymphoma” Cancer Pract 6: 195-7).A second example relates to antibodies which interfere with a criticalcomponent of tumor growth. Herceptin is an anti-HER-2 monoclonalantibody for treatment of metastatic breast cancer, and provides anexample of an antibody with this mechanism of action (Baselga et al.(1998) “Recombinant humanized anti-HER2 antibody (Herceptin) enhancesthe antitumor activity of paclitaxel and doxorubicin against HER2/neuoverexpressing human breast cancer xenografts [published erratum appearsin Cancer Res (1999) 59(8):2020], Cancer Res 58: 2825-31). A thirdexample relates to antibodies for delivery of cytotoxic compounds(toxins, radionuclides, etc.) directly to a tumor or other site ofinterest. For example, One application Mab is CYT-356, a 90Y-linkedantibody that targets radiation directly to prostate tumor cells (Deb etal. (1996) “Treatment of hormone-refractory prostate cancer with90Y-CYT-356 monoclonal antibody” Clin Cancer Res 2: 1289-97. A fourthapplication is antibody-directed enzyme prodrug therapy, where an enzymeco-localized to a tumor activates a systemically-administered pro-drugin the tumor vicinity. For example, an anti-Ep-CAM1 antibody linked tocarboxypeptidase A is being developed for treatment of colorectal cancer(Wolfe et al. (1999) “Antibody-directed enzyme prodrug therapy with theT268G mutant of human carboxypeptidase A1: in vitro and in vivo studieswith prodrugs of methotrexate and the thymidylate synthase inhibitorsGW1031 and GW1843” Bioconjug Chem 10: 38-48). Other Abs (e.g.,antagonists) are designed to specifically inhibit normal cellularfunctions for therapeutic benefit. An example is Orthoclone OKT3, ananti-CD3 MAb offered by Johnson and Johnson for reducing acute organtransplant rejection (Strate et al. (1990) “Orthoclone OKT3 asfirst-line therapy in acute renal allograft rejection” Transplant Proc22: 219-20. Another class of antibody products are agonists. These Mabsare designed to specifically enhance normal cellular functions fortherapeutic benefit. For example, Mab-based agonists of acetylcholinereceptors for neurotherapy are under development (Xie et al. (1997)“Direct demonstration of MuSK involvement in acetylcholine receptorclustering through identification of agonist ScFv” Nat. Biotechnol. 15:768-71. Any of these antibodies can be modified to include one or moreunnatural amino acid to enhance one or more therapeutic property(specificity, avidity, serum-half-life, etc.).

Another class of antibody products provide novel functions. The mainantibodies in this group are catalytic antibodies such as Ig sequencesthat have been engineered to mimic the catalytic abilities of enzymes(Wentworth and Janda (1998) “Catalytic antibodies” Curr Opin Chem Biol2: 138-44. For example, an interesting application involves using thecatalytic antibody mAb-15A10 to hydrolyze cocaine in vivo for addictiontherapy (Mets et al. (1998) “A catalytic antibody against cocaineprevents cocaine's reinforcing and toxic effects in rats” Proc Natl AcadSci USA 95: 10176-81). Catalytic antibodies can also be modified toinclude one or more unnatural amino acid to improve one or more propertyof interest.

Purifying Recombinant Proteins Comprising Unnatural Amino Acids

Proteins of the invention, e.g., proteins comprising unnatural aminoacids, antibodies to proteins comprising unnatural amino acids, etc.,can be purified, either partially or substantially to homogeneity,according to standard procedures known to and used by those of skill inthe art. Accordingly, polypeptides of the invention can be recovered andpurified by any of a number of methods well known in the art, including,e.g., ammonium sulfate or ethanol precipitation, acid or baseextraction, column chromatography, affinity column chromatography, anionor cation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, hydroxylapatite chromatography,lectin chromatography, gel electrophoresis and the like. Proteinrefolding steps can be used, as desired, in making correctly foldedmature proteins. High performance liquid chromatography (HPLC), affinitychromatography or other suitable methods can be employed in finalpurification steps where high purity is desired. In one embodiment,antibodies made against unnatural amino acids (or proteins comprisingunnatural amino acids) are used as purification reagents, e.g., foraffinity-based purification of proteins comprising one or more unnaturalamino acid(s). Once purified, partially or to homogeneity, as desired,the polypeptides are optionally used e.g., as assay components,therapeutic reagents or as immunogens for antibody production.

In addition to other references noted herein, a variety ofpurification/protein folding methods are well known in the art,including, e.g., those set forth in R. Scopes, Protein Purification,Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182:Guide to Protein Purification, Academic Press, Inc. N.Y. (1990); Sandana(1997) Bioseparation of Proteins, Academic Press, Inc.; Bollag et al.(1996) Protein Methods, 2nd Edition Wiley-Liss, NY; Walker (1996) TheProtein Protocols Handbook Humana Press, NJ, Harris and Angal (1990)Protein Purification Applications: A Practical Approach IRL Press atOxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993)Protein Purification Principles and Practice 3rd Edition SpringerVerlag, NY; Janson and Ryden (1998) Protein Purification: Principles,High Resolution Methods and Applications, Second Edition Wiley-VCH, NY;and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and thereferences cited therein.

As noted, those of skill in the art will recognize that, aftersynthesis, expression and/or purification, proteins can possess aconformation different from the desired conformations of the relevantpolypeptides. For example, polypeptides produced by prokaryotic systemsoften are optimized by exposure to chaotropic agents to achieve properfolding. During purification from, e.g., lysates derived from E. coli,the expressed protein is optionally denatured and then renatured. Thisis accomplished, e.g., by solubilizing the proteins in a chaotropicagent such as guanidine HCl.

In general, it is occasionally desirable to denature and reduceexpressed polypeptides and then to cause the polypeptides to re-foldinto the preferred conformation. For example, guanidine, urea, DTT, DTE,and/or a chaperonin can be added to a translation product of interest.Methods of reducing, denaturing and renaturing proteins are well knownto those of skill in the art (see, the references above, and Debinski,et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan(1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal.Biochem., 205: 263-270). Debinski, et al., for example, describe thedenaturation and reduction of inclusion body proteins in guanidine-DTE.The proteins can be refolded in a redox buffer containing, e.g.,oxidized glutathione and L-arginine. Refolding reagents can be flowed orotherwise moved into contact with the one or more polypeptide or otherexpression product, or vice-versa.

Nucleic Acid and Polypeptide Sequence Variants

As described above and below, the invention provides for nucleic acidpolynucleotide sequences and polypeptide amino acid sequences, e.g.,O-tRNAs and O-RSs, and, e.g., compositions and methods comprising saidsequences. Examples of said sequences, e.g., O-tRNAs and O-RSs aredisclosed herein. However, one of skill in the art will appreciate thatthe invention is not limited to those sequences disclosed herein, e.g.,the Examples. One of skill will appreciate that the present inventionalso provides many unrelated sequences with the functions describedherein, e.g., encoding an O-tRNA or an O-RS.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences, wherein the variants hybridize toat least one disclosed sequence, are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques, are alsoincluded in the invention.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence which encodes an amino acid. Similarly,“conservative amino acid substitutions,” in one or a few amino acids inan amino acid sequence are substituted with different amino acids withhighly similar properties, are also readily identified as being highlysimilar to a disclosed construct. Such conservative variations of eachdisclosed sequence are a feature of the present invention.

“Conservative variations” of a particular nucleic acid sequence refersto those nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences. One of skillwill recognize that individual substitutions, deletions or additionswhich alter, add or delete a single amino acid or a small percentage ofamino acids (typically less than 5%, more typically less than 4%, 2% or1%) in an encoded sequence are “conservatively modified variations”where the alterations result in the deletion of an amino acid, additionof an amino acid, or substitution of an amino acid with a chemicallysimilar amino acid. Thus, “conservative variations” of a listedpolypeptide sequence of the present invention include substitutions of asmall percentage, typically less than 5%, more typically less than 2% or1%, of the amino acids of the polypeptide sequence, with aconservatively selected amino acid of the same conservative substitutiongroup. Finally, the addition of sequences which do not alter the encodedactivity of a nucleic acid molecule, such as the addition of anon-functional sequence, is a conservative variation of the basicnucleic acid.

Conservative substitution tables providing functionally similar aminoacids are well known in the art. The following sets forth example groupswhich contain natural amino acids that include “conservativesubstitutions” for one another.

Conservative Substitution Groups 1 Alanine (A) Serine (S) Threonine (T)2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M)Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of theinvention, including conservative variations of nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. In addition,target nucleic acids which hybridize to the nucleic acids represented bySEQ ID NO:1-34 under high, ultra-high and ultra-ultra high stringencyconditions are a feature of the invention. Examples of such nucleicacids include those with one or a few silent or conservative nucleicacid substitutions as compared to a given nucleic acid sequence.

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at lest ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 5×-10× as high as that observed for hybridization toany of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes part I chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, New York), aswell as in Ausubel, supra. Hames and Higgins (1995) Gene Probes 1 IRLPress at Oxford University Press, Oxford, England, (Hames and Higgins 1)and Hames and Higgins (1995) Gene Probes 2 IRL Press at OxfordUniversity Press, Oxford, England (Hames and Higgins 2) provide detailson the synthesis, labeling, detection and quantification of DNA and RNA,including oligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions is a 0.2×SSC wash at65° C. for 15 minutes (see, Sambrook, supra for a description of SSCbuffer). Often the high stringency wash is preceded by a low stringencywash to remove background probe signal. An example low stringency washis 2×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 5× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra. and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determininghighly stringent hybridization and wash conditions, the hybridizationand wash conditions are gradually increased (e.g., by increasingtemperature, decreasing salt concentration, increasing detergentconcentration and/or increasing the concentration of organic solventssuch as formalin in the hybridization or wash), until a selected set ofcriteria are met. For example, the hybridization and wash conditions aregradually increased until a probe binds to a perfectly matchedcomplementary target with a signal to noise ratio that is at least 5× ashigh as that observed for hybridization of the probe to an unmatchedtarget.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids. A target nucleic acid which hybridizes to a probe undersuch conditions, with a signal to noise ratio of at least ½ that of theperfectly matched complementary target nucleic acid is said to bind tothe probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids. A target nucleic acid which hybridizesto a probe under such conditions, with a signal to noise ratio of atleast ½ that of the perfectly matched complementary target nucleic acidis said to bind to the probe under ultra-ultra-high stringencyconditions.

Nucleic acids which do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Unique Subsequences

In one aspect, the invention provides a nucleic acid which comprises aunique subsequence in a nucleic acid selected from the sequences ofO-tRNAs and O-RSs disclosed herein. The unique subsequence is unique ascompared to a nucleic acid corresponding to any known O-tRNA or O-RSnucleic acid sequence. Alignment can be performed using, e.g., BLAST setto default parameters. Any unique subsequence is useful, e.g., as aprobe to identify the nucleic acids of the invention.

Similarly, the invention includes a polypeptide which comprises a uniquesubsequence in a polypeptide selected from the sequences of O-RSsdisclosed herein. Here, the unique subsequence is unique as compared toa polypeptide corresponding to any of known polypeptide sequence.

The invention also provides for target nucleic acids which hybridizesunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from thesequences of O-RSs wherein the unique subsequence is unique as comparedto a polypeptide corresponding to any of the control polypeptides (e.g.,parental sequences from which synthetases of the invention were derived,e.g., by mutation). Unique sequences are determined as noted above.

Sequence Comparison, Identity, and Homology

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs encoding an O-tRNA or O-RS, or theamino acid sequence of an O-RS) refers to two or more sequences orsubsequences that have at least about 60%, preferably 80%, mostpreferably 90-95% nucleotide or amino acid residue identity, whencompared and aligned for maximum correspondence, as measured using asequence comparison algorithm or by visual inspection. Such“substantially identical” sequences are typically considered to be“homologous,” without reference to actual ancestry. Preferably, the“substantial identity” exists over a region of the sequences that is atleast about 50 residues in length, more preferably over a region of atleast about 100 residues, and most preferably the sequences aresubstantially identical over at least about 150 residues, or over thefull length of the two sequences to be compared.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (on the world wide web(www.) at ncbi.nlm.nih on the government domain (.gov/)). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are then extended inboth directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the invention provide a variety of newpolypeptide sequences (e.g., comprising unnatural amino acids in thecase of proteins synthesized in the translation systems herein, or,e.g., in the case of the novel synthetases herein, novel sequences ofstandard amino acids), the polypeptides also provide new structuralfeatures which can be recognized, e.g., in immunological assays. Thegeneration of antisera which specifically bind the polypeptides of theinvention, as well as the polypeptides which are bound by such antisera,are a feature of the invention.

For example, the invention includes synthetase proteins thatspecifically bind to or that are specifically immunoreactive with anantibody or antisera generated against an immunogen comprising an aminoacid sequence selected from one or more of (SEQ ID NO:35-66. Toeliminate cross-reactivity with other homologues, the antibody orantisera is subtracted with available synthetases, such as the wild-typeMethanococcus jannaschii (M. jannaschii) tyrosyl synthetase (TyrRS).Where the wild-type Methanococcus jannaschii (M. jannaschii) tyrosylsynthetase (TyrRS) corresponds to a nucleic acid, a polypeptide encodedby the nucleic acid is generated and used for antibody/antiserasubtraction purposes.

In one typical format, the immunoassay uses a polyclonal antiserum whichwas raised against one or more polypeptide comprising one or more of thesequences corresponding to one or more of SEQ ID NO:35-66) or asubstantial subsequence thereof (i.e., at least about 30% of the fulllength sequence provided). The set of potential polypeptide immunogensderived from SEQ ID NO:35-66) are collectively referred to below as “theimmunogenic polypeptides.” The resulting antisera is optionally selectedto have low cross-reactivity against the control synthetase homologuesand any such cross-reactivity is removed, e.g., by immunoabsorbtion,with one or more of the control synthetase homologues, prior to use ofthe polyclonal antiserum in the immunoassay.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein can be produced in arecombinant cell. An inbred strain of mice (used in this assay becauseresults are more reproducible due to the virtual genetic identity of themice) is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity.Additional references and discussion of antibodies is also found hereinand can be applied here to defining polypeptides by immunoreactivity).Alternatively, one or more synthetic or recombinant polypeptide derivedfrom the sequences disclosed herein is conjugated to a carrier proteinand used as an immunogen.

Polyclonal sera are collected and titered against the immunogenicpolypeptide in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control synthetase polypeptidesto produce subtracted pooled titered polyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for crossreactivity against the control homologues in a comparative immunoassay.In this comparative assay, discriminatory binding conditions aredetermined for the subtracted titered polyclonal antisera which resultin at least about a 5-10 fold higher signal to noise ratio for bindingof the titered polyclonal antisera to the immunogenic synthetase ascompared to binding to the control synthetase homologues. That is, thestringency of the binding reaction is adjusted by the addition ofnon-specific competitors such as albumin or non-fat dry milk, and/or byadjusting salt conditions, temperature, and/or the like. These bindingconditions are used in subsequent assays for determining whether a testpolypeptide (a polypeptide being compared to the immunogenicpolypeptides and/or the control polypeptides) is specifically bound bythe pooled subtracted polyclonal antisera. In particular, testpolypeptides which show at least a 2-5× higher signal to noise ratiothan the control synthetase homologues under discriminatory bindingconditions, and at least about a ½ signal to noise ratio as compared tothe immunogenic polypeptide(s), shares substantial structural similaritywith the immunogenic polypeptide as compared to known synthetases, andis, therefore a polypeptide of the invention.

In another example, immunoassays in the competitive binding format areused for detection of a test polypeptide. For example, as noted,cross-reacting antibodies are removed from the pooled antisera mixtureby immunoabsorbtion with the control polypeptides. The immunogenicpolypeptide(s) are then immobilized to a solid support which is exposedto the subtracted pooled antisera. Test proteins are added to the assayto compete for binding to the pooled subtracted antisera. The ability ofthe test protein(s) to compete for binding to the pooled subtractedantisera as compared to the immobilized protein(s) is compared to theability of the immunogenic polypeptide(s) added to the assay to competefor binding (the immunogenic polypeptides compete effectively with theimmobilized immunogenic polypeptides for binding to the pooledantisera). The percent cross-reactivity for the test proteins iscalculated, using standard calculations.

In a parallel assay, the ability of the control proteins to compete forbinding to the pooled subtracted antisera is optionally determined ascompared to the ability of the immunogenic polypeptide(s) to compete forbinding to the antisera. Again, the percent cross-reactivity for thecontrol polypeptides is calculated, using standard calculations. Wherethe percent cross-reactivity is at least 5-10× as high for the testpolypeptides as compared to the control polypeptides and or where thebinding of the test polypeptides is approximately in the range of thebinding of the immunogenic polypeptides, the test polypeptides are saidto specifically bind the pooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in acompetitive binding immunoassay as described herein to compare any testpolypeptide to the immunogenic and/or control polypeptide(s). In orderto make this comparison, the immunogenic, test and control polypeptidesare each assayed at a wide range of concentrations and the amount ofeach polypeptide required to inhibit 50% of the binding of thesubtracted antisera to, e.g., an immobilized control, test orimmunogenic protein is determined using standard techniques. If theamount of the test polypeptide required for binding in the competitiveassay is less than twice the amount of the immunogenic polypeptide thatis required, then the test polypeptide is said to specifically bind toan antibody generated to the immunogenic protein, provided the amount isat least about 5-10× as high as for the control polypeptide.

As an additional determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptides) until little or no binding of theresulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunosorbtion is detectable.This fully immunosorbed antisera is then tested for reactivity with thetest polypeptide. If little or no reactivity is observed (i.e., no morethan 2× the signal to noise ratio observed for binding of the fullyimmunosorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic protein.

General Molecular Biology Techniques

General texts which describe molecular biological techniques includeBerger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger);Sambrook et al., Molecular Cloning—A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989(“Sambrook”) and Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 1999) (“Ausubel”)). These texts describe mutagenesis, the use ofvectors, promoters and many other relevant topics related to, e.g., thegeneration of genes that include selector codons for production ofproteins that include unnatural amino acids, orthogonal tRNAs,orthogonal synthetases, and pairs thereof.

Various types of mutagenesis are used in the present invention, e.g., toinsert selector codons that encode unnatural amino acids in a protein.They include but are not limited to site-directed, random pointmutagenesis, homologous recombination (DNA shuffling), mutagenesis usinguracil containing templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,are also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like.

The above texts and examples found herein describe these procedures.Additional information is found in the following publications andreferences cited within: Ling et al., Approaches to DNA mutagenesis: anoverview, Anal Biochem. 254(2): 157-178 (1997); Dale et al.,Oligonucleotide-directed random mutagenesis using the phosphorothioatemethod, Methods Mol. Biol. 57:369-374 (1996); Smith, In vitromutagenesis, Ann. Rev. Genet. 19:423-462 (1985); Botstein & Shortle,Strategies and applications of in vitro mutagenesis, Science229:1193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)) (1987); Kunkel, Rapidand efficient site-specific mutagenesis without phenotypic selection,Proc. Natl. Acad. Sci. USA 82:488-492 (1985); Kunkel et al., Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trprepressors with new DNA-binding specificities, Science 242:240-245(1988); Methods in Enzymol. 100: 468-500 (1983); Methods in Enzymol.154: 329-350 (1987); Zoller & Smith, Oligonucleotide-directedmutagenesis using M13-derived vectors: an efficient and generalprocedure for the production of point mutations in any DNA fragment,Nucleic Acids Res. 10:6487-6500 (1982); Zoller & Smith,Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors, Methods in Enzymol. 100:468-500 (1983); Zoller & Smith,Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template, Methods inEnzymol. 154:329-350 (1987); Taylor et al., The use ofphosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA, Nucl. Acids Res. 13: 8749-8764 (1985); Taylor et al., Therapid generation of oligonucleotide-directed mutations at high frequencyusing phosphorothioate-modified DNA, Nucl. Acids Res. 13: 8765-8787(1985); Nakamaye & Eckstein, Inhibition of restriction endonuclease NciI cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, Nucl. Acids Res. 14: 9679-9698(1986); Sayers et al., Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis, Nucl. Acids Res. 16:791-802(1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl. AcidsRes. 16: 803-814; Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl. Acids Res. 12:9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed constructionof mutations via gapped duplex DNA, Methods in Enzymol. 154:350-367(1987); Kramer et al., Improved enzymatic in vitro reactions in thegapped duplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl. Acids Res. 16: 7207 (1988); Fritz et al.,Oligonucleotide-directed construction of mutations: a gapped duplex DNAprocedure without enzymatic reactions in vitro, Nucl. Acids Res. 16:6987-6999 (1988); Kramer et al., Point Mismatch Repair, Cell 38:879-887(1984); Carter et al., Improved oligonucleotide site-directedmutagenesis using M13 vectors, Nucl. Acids Res. 13: 4431-4443 (1985);Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors,Methods in Enzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Useof oligonucleotides to generate large deletions, Nucl. Acids Res. 14:5115 (1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil. Trans. R. Soc.Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloningof a gene coding for the ribonuclease S protein, Science 223: 1299-1301(1984); Sakamar and Khorana, Total synthesis and expression of a genefor the a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin), Nucl. Acids Res. 14: 6361-6372 (1988); Wells etal., Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites, Gene 34:315-323 (1985); Grundströmet al., Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’gene synthesis, Nucl. Acids Res. 13: 3305-3316 (1985); Mandecki,Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis, Proc. Natl.Acad. Sci. USA, 83:7177-7181 (1986); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Sieber, et al., Nature Biotechnology, 19:456-460 (2001). W. P. C.Stemmer, Nature 370, 389-91 (1994); and, I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995). Additional details on many of theabove methods can be found in Methods in Enzymology Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

The present invention also relates to host cells and organisms for thein vivo incorporation of an unnatural amino acid via orthogonal tRNA/RSpairs. Host cells are genetically engineered (e.g., transformed,transduced or transfected) with the vectors of this invention, which canbe, for example, a cloning vector or an expression vector. The vectorcan be, for example, in the form of a plasmid, a bacterium, a virus, anaked polynucleotide, or a conjugated polynucleotide. The vectors areintroduced into cells and/or microorganisms by standard methodsincluding electroporation (From et al., Proc. Natl. Acad. Sci. USA 82,5824 (1985), infection by viral vectors, high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.,Nature 327, 70-73 (1987)).

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, screeningsteps, activating promoters or selecting transformants. These cells canoptionally be cultured into transgenic organisms.

Several well-known methods of introducing target nucleic acids intobacterial cells are available, any of which can be used in the presentinvention. These include: fusion of the recipient cells with bacterialprotoplasts containing the DNA, electroporation, projectile bombardment,and infection with viral vectors (discussed further, below), etc.Bacterial cells can be used to amplify the number of plasmids containingDNA constructs of this invention. The bacteria are grown to log phaseand the plasmids within the bacteria can be isolated by a variety ofmethods known in the art (see, for instance, Sambrook). In addition, aplethora of kits are commercially available for the purification ofplasmids from bacteria, (see, e.g., EasyPrep™, FlexiPrep™, both fromPharmacia Biotech; StrataClean™, from Stratagene; and, QIAprep™ fromQiagen). The isolated and purified plasmids are then further manipulatedto produce other plasmids, used to transfect cells or incorporated intorelated vectors to infect organisms. Typical vectors containtranscription and translation terminators, transcription and translationinitiation sequences, and promoters useful for regulation of theexpression of the particular target nucleic acid. The vectors optionallycomprise generic expression cassettes containing at least oneindependent terminator sequence, sequences permitting replication of thecassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors)and selection markers for both prokaryotic and eukaryotic systems.Vectors are suitable for replication and integration in prokaryotes,eukaryotes, or preferably both. See, Giliman & Smith, Gene 8:81 (1979);Roberts, et al., Nature, 328:731 (1987); Schneider, B., et al., ProteinExpr. Purif 6435:10 (1995); Ausubel, Sambrook, Berger (all supra). Acatalogue of Bacteria and Bacteriophages useful for cloning is provided,e.g., by the ATCC, e.g., The ATCC Catalogue of Bacteria andBacteriophage (1992) Gherna et al. (eds) published by the ATCC.Additional basic procedures for sequencing, cloning and other aspects ofmolecular biology and underlying theoretical considerations are alsofound in Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, NY.

Other useful references, e.g. for cell isolation and culture (e.g., forsubsequent nucleic acid isolation) include Freshney (1994) Culture ofAnimal Cells, a Manual of Basic Technique, third edition, Wiley-Liss,New York and the references cited therein; Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla.

In addition, essentially any nucleic acid (and virtually any labelednucleic acid, whether standard or non-standard) can be custom orstandard ordered from any of a variety of commercial sources, such asThe Midland Certified Reagent Company (mcrc at (@) oligos on thecommercial domain (.com), The Great American Gene Company (on the worldwide web (www) at genco on the commercial domain (.com)), ExpressGenInc. (on the world wide web (www) at expressgen on the commercialdomain(.com)), Operon Technologies Inc. (Alameda, Calif.) and manyothers.

Pharmaceutical Compositions

The proteins, e.g., polypeptides, peptides, etc., of the invention(e.g., comprising one or more unnatural amino acid) are optionallyemployed for therapeutic uses, e.g., in combination with a suitablepharmaceutical carrier. Such compositions, e.g., comprise atherapeutically effective amount of the compound, and a pharmaceuticallyacceptable carrier or excipient. Such a carrier or excipient includes,but is not limited to, saline, buffered saline, dextrose, water,glycerol, ethanol, and/or combinations thereof. The formulation is madeto suit the mode of administration. In general, methods of administeringproteins are well known in the art and can be applied to administrationof the polypeptides of the invention.

Therapeutic compositions comprising one or more polypeptide of theinvention are optionally tested in one or more appropriate in vitroand/or in vivo animal models of disease, to confirm efficacy, tissuemetabolism, and to estimate dosages, according to methods well known inthe art. In particular, dosages can be initially determined by activity,stability or other suitable measures of unnatural herein to naturalamino acid homologues (e.g., comparison of an EPO modified to includeone or more unnatural amino acids to a natural amino acid EPO), i.e., ina relevant assay.

Administration is by any of the routes normally used for introducing amolecule into ultimate contact with blood or tissue cells. The unnaturalamino acid polypeptides of the invention are administered in anysuitable manner, optionally with one or more pharmaceutically acceptablecarriers. Suitable methods of administering such polypeptides in thecontext of the present invention to a patient are available, and,although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective action or reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention.

Polypeptide compositions can be administered by a number of routesincluding, but not limited to: oral, intravenous, intraperitoneal,intramuscular, transdermal, subcutaneous, topical, sublingual, or rectalmeans. Unnatural amino acid polypeptide compositions can also beadministered via liposomes. Such administration routes and appropriateformulations are generally known to those of skill in the art.

The unnatural amino acid polypeptide, alone or in combination with othersuitable components, can also be made into aerosol formulations (i.e.,they can be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations of packaged nucleic acid can be presented in unit-doseor multi-dose sealed containers, such as ampules and vials.

Parenteral administration and intravenous administration are preferredmethods of administration. In particular, the routes of administrationalready in use for natural amino acid homologue therapeutics (e.g.,those typically used for EPO, GCSF, GMCSF, IFNs, interleukins,antibodies, and/or any other pharmaceutically delivered protein), alongwith formulations in current use, provide preferred routes ofadministration and formulation for the unnatural amino acids of theinvention.

The dose administered to a patient, in the context of the presentinvention, is sufficient to effect a beneficial therapeutic response inthe patient over time, or, e.g., to inhibit infection by a pathogen, orother appropriate activity, depending on the application. The dose isdetermined by the efficacy of the particular vector, or formulation, andthe activity, stability or serum half-life of the unnatural amino acidpolypeptide employed and the condition of the patient, as well as thebody weight or surface area of the patient to be treated. The size ofthe dose is also determined by the existence, nature, and extent of anyadverse side-effects that accompany the administration of a particularvector, formulation, or the like in a particular patient.

In determining the effective amount of the vector or formulation to beadministered in the treatment or prophylaxis of disease (e.g., cancers,inherited diseases, diabetes, AIDS, or the like), the physicianevaluates circulating plasma levels, formulation toxicities, progressionof the disease, and/or where relevant, the production of anti-unnaturalamino acid polypeptide antibodies.

The dose administered, e.g., to a 70 kilogram patient are typically inthe range equivalent to dosages of currently-used therapeutic proteins,adjusted for the altered activity or serum half-life of the relevantcomposition. The vectors of this invention can supplement treatmentconditions by any known conventional therapy, including antibodyadministration, vaccine administration, administration of cytotoxicagents, natural amino acid polypeptides, nucleic acids, nucleotideanalogues, biologic response modifiers, and the like.

For administration, formulations of the present invention areadministered at a rate determined by the LD-50 of the relevantformulation, and/or observation of any side-effects of the unnaturalamino acids at various concentrations, e.g., as applied to the mass andoverall health of the patient. Administration can be accomplished viasingle or divided doses.

If a patient undergoing infusion of a formulation develops fevers,chills, or muscle aches, he/she receives the appropriate dose ofaspirin, ibuprofen, acetaminophen or other pain/fever controlling drug.Patients who experience reactions to the infusion such as fever, muscleaches, and chills are premedicated 30 minutes prior to the futureinfusions with either aspirin, acetaminophen, or, e.g., diphenhydramine.Meperidine is used for more severe chills and muscle aches that do notquickly respond to antipyretics and antihistamines. Cell infusion isslowed or discontinued depending upon the severity of the reaction.

EXAMPLES Example 1 In Vivo Incorporation of O-methyl-L-tyrosine

An orthogonal tRNA/synthetase pair in E. coli can be generated byimporting a pair from a different organism, if cross-speciesaminoacylation is inefficient (Y. Kwok, J. T. Wong, Can. J. Biochem. 58,213-8 (1980)), and the anticodon loop is not a key determinant ofsynthetase recognition. One such candidate pair is the tyrosyltRNA/synthetase pair of Methanococcus jannaschii (M. jannaschii), anarchaebacterium whose tRNA identity elements differ from those of E.coli tRNATyr, and whose tyrosyl synthetase (TyrRS) lacks an anticodonloop binding domain (B. A. Steer, P. Schimmel, J. Biol. Chem. 274,35601-6 (1999)). In addition, the M. jannaschii TyrRS does not have anediting mechanism (H. Jakubowski, E. Goldman, Microbiol. Rev. 56, 412-29(1992)), and therefore should not proofread an unnatural amino acidligated to the tRNA.

It has been shown that an amber suppressor tRNA derived from the M.jannaschii tRNA^(Tyr) is not efficiently aminoacylated by the E. colisynthetases, but functions efficiently in protein translation in E. coli(L. Wang, T. J. Magliery, D. R. Liu, P. G. Schultz, J. Am. Chem. Soc.122, 5010-1 (2000)). Moreover, the M. jannaschii TyrRS is orthogonal toE. coli tRNAs (B. A. Steer, P. Schimmel, J. Biol. Chem. 274, 35601-6(1999)), but still efficiently aminoacylates its own suppressortRNA_(CUA) ^(Tyr) (L. Wang, T. J. Magliery, D. R. Liu, P. G. Schultz, J.Am. Chem. Soc. 122, 5010-1 (2000)). Thus, the M. jannaschii tRNA_(CUA)^(Tyr)/TyrRS functions as an orthogonal pair, and can efficientlyincorporate tyrosine in response to the amber codon, UAG, in E. coli.

To further reduce recognition of this orthogonal tRNA by E. colisynthetases, mutagenesis and selection scheme was performed. Foradditional details, see U.S. patent application Ser. No. 10/126,931,titled “Methods and Compositions for the production of orthogonaltRNA-tRNA synthetase pairs,” the disclosure of which is incorporated inits entirety.

Briefly, eleven nucleotides of the M. jannaschii tRNA_(CUA) ^(Tyr) thatdo not interact directly with the M. jannaschii TyrRS were randomlymutated to afford a suppressor tRNA library. This tRNA library waspassed through a negative selection that removes tRNAs that areaminoacylated by E. coli synthetases, followed by a positive selectionfor tRNAs that are efficiently aminoacylated by M. jannaschii TyrRS. Theorthogonality of the resulting suppressor tRNAs was tested by an in vivocomplementation assay, based on suppression of an amber stop codon at anonessential position (Ala184) of the TEM-1 beta-lactamase gene carriedon plasmid pBLAM. Aminoacylation of a transformed suppressor tRNA by anyendogenous E. coli synthetase results in cell growth in the presence ofampicillin. E. coli transformed with the M. jannaschii tRNA_(CUA) ^(Tyr)and pBLAM survive at 55.5 micrograms/mL ampicillin. When the best mutantsuppressor tRNA (mtRNA_(CUA) ^(Tyr)) selected from the library wasexpressed, cells survived at only 12.4 micrograms/mL ampicillin. Themutant suppressor mtRNA_(CUA) ^(Tyr) contained the following nucleotidesubstitutions: C17A, U17aG, U20C, G37A, and U47G. For comparison, cellswith pBLAM only (in the absence of any suppressor tRNA) survive at 9.7micrograms/mL ampicillin. When the M. jannaschii TyrRS is coexpressedwith this mtRNA_(CUA) ^(Tyr), cells survive at 436 micrograms/mLampicillin. Thus, the mtRNA_(CUA) ^(Tyr) is a poorer substrate for theendogenous synthetases than the M. jannaschii tRNA_(CUA) ^(Tyr) and isstill aminoacylated efficiently by the M. jannaschii TyrRS.

To alter the amino acid specificity of the orthogonal M. jannaschiiTyrRS so that it charges the mtRNA_(CUA) ^(Tyr) with a desired unnaturalamino acid, a library of TyrRS mutants was generated and screened. Basedon the crystal structure of the homologous TyrRS from Bacillusstearothermophilus (P. Brick, T. N. Bhat, D. M. Blow, J. Mol. Biol. 208,83-98 (1988)), five residues (Tyr32, Glu107, Asp158, Ile159 and Leu162)in the active site of M. jannaschii TyrRS, which are within 6.5 Å of thepara position of the aryl ring of bound tyrosine were mutated.Corresponding residues from a mutant M. jannaschii TyrRS (mutTyr RS, foraminoacylation with O-methyl-L-tyrosine) are Tyr³² (Tyr³⁴), Glu¹⁰⁷(Asn¹²³), Asp¹⁵⁸ (Asp¹⁷⁶), Ile¹⁵⁹ (Phe¹⁷⁷), and Leu¹⁶² (Leu¹⁸⁰) with B.stearothermophilus TyrRS residues in parenthesis.

As described in more detail below, these residues were all initiallymutated to alanine to generate an Ala5 TyrRS, which is unable to chargethe mtRNA_(CUA) ^(Tyr) with tyrosine. This mutant Ala5 TyrRS was used asa template to generate a library of TyrRS mutants in which the fiveresidues were randomly mutated by PCR mutagenesis with dopedoligonucleotides.

The M. jannaschii TyrRS gene was expressed under the control of E. coliGlnRS promoter and terminator in plasmid pBK-JYRS, a pBR322 derivedplasmid with kanamycin resistance. Residues Tyr32, Glu107, Asp158,Ile159 and Leu162 were substituted with Ala by site-directed mutagenesisto afford plasmid pBK-JYA5. Oligonucleotides LW1575′-GGAATTCCATATGGACGAATTTGAAATG-3′(SEQ ID NO:69), LW164 5′-GTATTTTACCACTTGGTTCAAAACCTATMNNAGCAGATTTTTCATCTTTTTTTCATCTTT TTTTAAAAC-3′(SEQID NO:70), LW159 5′-TAGGTTTTGAACCAAGTGGTAAAATAC-3′ (SEQ ID NO:71), LW1655′-CATTCAGTGTATAATCCTTATCAAGCTGGAAMNNACTTCCATAA ACATATTTTGCCTTTAAC-3′(SEQ ID NO:72), LW161 5′-TCCAGCTTGATAAGGATTATACA CTGAATG-3′ (SEQ IDNO:73), LW167 5′-CATCCCTCCAACTGCAACATCAACGCCMNNATAATGMNNMNNATTAACCTGCATTATTGGATAGATAAC-3′ (SEQ ID NO:74), LW163 5′-GCGTTGATGTTGCAGTTGGAGGGATG-3′ (SEQ ID NO:75), and LW105 5′-AAACTGCAGTTATAATCTCTTTCTAATTGGCTC-3′ (SEQ ID NO:76) with NNK (N=A+T+G+C, K=G+T, andM=C+A (Operon, Alameda, Calif.) at the mutation sites were used for PCRamplification of the Ala5 TyrRS mutant (pBK-JYA5) and ligated back intothe NdeI-PstI-digested pBK-JYA5 to afford the TyrRS library. The ligatedvectors were transformed into E. coli DH10B competent cells to yield alibrary of 1.6×10⁹ colony forming unit (cfu). The TyrRS genes from 40randomly picked colonies were sequenced to confirm that there was nobase bias at the randomized NNK positions and no other unexpectedmutations. The library was amplified by maxiprep, and supercoiled DNAwas used to transform the selection strain pYC-J17.

A positive selection was then applied that is based on suppression of anamber stop codon at a nonessential position (Asp112) in thechloramphenicol acetyltransferase (CAT) gene (M. Pastrnak, T. J.Magliery, P. G. Schultz, Helvetica Chimica Acta. 83, 2277-86 (2000)).Cells were grown in media containing the unnatural amino acid andselected for their survival in the presence of various concentration ofchloramphenicol. If a mutant TyrRS charges the orthogonal mtRNA_(CUA)^(Tyr) with any amino acid, either natural or unnatural, the cellproduces CAT and survives.

The surviving cells were then grown in the presence of chloramphenicoland in the absence of the unnatural amino acid. Those cells that did notsurvive, i.e., which encode mutant TyrRSs that charge the orthogonalmtRNA_(CUA) ^(Tyr) with an unnatural amino acid, were isolated from areplica plate supplemented with the unnatural amino acid. The mutantTyrRS genes were isolated from these cells, recombined in vitro by DNAshuffling, and transformed back into E. coli for further rounds ofselection.

Seven tyrosine analogues with different functional groups at the paraposition of the aryl ring (acetyl, amino, carboxyl, isopropyl, methyl,O-methyl and nitro) were used individually in the selections that wereperformed as follows.

The gene encoding mtRNA_(CUA) ^(Tyr) under the control of the lpppromoter and rrnC terminator was inserted into plasmid pACMD112TAG (apACYC184 plasmid with a TAG stop codon replacing Asp112 in its CAT gene(M. Pastrnak, T. J. Magliery, P. G. Schultz, Helvetica Chimica Acta. 83,2277-86 (2000))) to afford plasmid pYC-J17. Supercoiled DNA encoding theTyrRS library was transformed into E. coli DH10B competent cellscontaining pYC-J17 to yield a library of size greater than 3×10⁹ cfu,ensuring complete coverage of the original library. Cells were thenplated on minimal media plates containing 1% glycerol and 0.3 mM leucine(GMML) with 17 micrograms/mL tetracycline(Tet), 25 micrograms/mLkanamycin (Kan), 50 micrograms/mL of chloramphenicol (Cm), and 1 mMunnatural amino acid. After incubation at 37° C. for 44 hours, colonieson plates supplied with O-methyl-L-tyrosine were pooled, plasmids wereisolated and retransformed into E. coli DH10B competent cells containingpYC-J17, and the transformed cells were positively selected on 50micrograms/mL of Cm. Colonies (96) were individually picked from theplate, diluted into 100 mL of liquid GMML media, and streaked onto twosets of Kan/Tet GMML plates with various concentration of Cm. NoO-methyl-L-tyrosine was added to plate set 1 and the concentration of Cmwas varied from 10-25 micrograms/mL; plate set 2 contained 1 mMO-methyl-L-tyrosine and 50 micrograms/mL of Cm. Replicates of coloniesthat did not grow on 15 micrograms/mL of Cm in plate set 1 were pickedfrom plate set 2. Plasmids containing the TyrRS gene were purified andrecombined in vitro by DNA shuffling using Stemmer's protocol (W. P. C.Stemmer, Nature 370, 389-91 (1994)) with the exception of 10 mM Mn2+instead of Mg2+ in the fragmentation reaction (I. A. Lorimer, I. Pastan,Nucleic Acids Res. 23, 3067-8 (1995)). The library was then religatedinto predigested pBK-JYA5 vector to afford a second generation TyrRSlibrary with a typical size of 8×108 to 3×109 cfu. Thirty randomlyselected members from the library were sequenced. The mutagenic rateintroduced by DNA shuffling was 0.35%. This library was transformed intothe selection strain for the next round of selection followed byshuffling. The concentration of Cm in the positive selection and inplate set 2 was raised to 80 micrograms/mL for the second round and 120micrograms/mL for the third round; the concentration of Cm in plate set1 was unchanged. After three rounds of DNA shuffling, colonies began togrow on 20-25 micrograms/mL Cm in plate set 1, indicating that the TyrRSmutants were accepting natural amino acids as substrates. Therefore, thebest clone selected after two rounds of DNA shuffling was characterizedin detail.

Following the two rounds of selection and DNA shuffling, a clone (mutantTyrRS (LWJ16)) was evolved whose survival in chloramphenicol wasdependent on the addition of 1 mM O-methyl-L-tyrosine to the growthmedia. In the absence of O-methyl-L-tyrosine, cells harboring the mutantTyrRS (LWJ16) were not viable on minimal media plates containing 1%glycerol, 0.3 mM leucine (GMML), and 15 micrograms/mL ofchloramphenicol. Cells were able to grow on GMML plates with 125micrograms/mL chloramphenicol in the presence of 1 mMO-methyl-L-tyrosine. Similar results were obtained in liquid GMML. As acontrol, cells with the mtRNA_(CUA) ^(Tyr) and the inactive Ala5 TyrRSdid not survive at the lowest concentration of chloramphenicol used,either in the presence or absence of 1 mM O-methyl-L-tyrosine. Thisindicates that the growth of cells in chloramphenicol relies on theexpression of the mutant TyrRS (LWJ16) and is not a simple nutritionaleffect of O-methyl-L-tyrosine. Addition of 1 mM O-methyl-L-tyrosineitself does not significantly affect the growth rate of E. coli.

To further demonstrate that the observed phenotype is due to thesite-specific incorporation of O-methyl-L-tyrosine by the orthogonalmtRNA_(CUA) ^(Tyr)/mutant TyrRS (LWJ16) pair in response to an amberstop codon, an O-methyl-L-tyrosine mutant of dihydrofolate reductase(DHFR) was generated and characterized. The third codon of the E. coliDHFR gene (a permissive site) was mutated to TAG and a C-terminal His₆tag was added in order to separate the mutant protein from endogenous E.coli DHFR. As a control, the mtRNA_(CUA) ^(Tyr) was coexpressed with thewild type M. jannaschii TyrRS, resulting in efficient suppression of thenonsense codon in DHFR with tyrosine. See FIG. 2. When the mutant TyrRS(LWJ16) was expressed in the presence of mtRNA_(CUA) ^(Tyr) and 1 mMO-methyl-L-tyrosine in liquid GMML growth media, full length DHFR wasalso produced and could be purified by Ni affinity chromatography withan isolated yield of 2 mg/liter.

The yield of purified protein is approximately 26 fold lower in liquidGMML media compare to 2YT rich media. For example, when the mtRNA_(CUA)^(Tyr) and wild type M. jannaschii TyrRS are coexpressed, the yield ofDHFR is 67 mg/L in 2YT and 2.6 mg/L in liquid GMML.

In the absence of either O-methyl-L-tyrosine, mtRNA_(CUA) ^(Tyr) ormutant TyrRS (LWJ16), no DHFR (<0.1% by densitometry) was observed byanalysis with SDS-polyacrylamide gel electrophoresis and silverstaining. See FIG. 2. Western analysis further demonstrated that notrace amount of DHFR was produced in the absence of either mtRNA_(CUA)^(Tyr), mutant TyrRS (LWJ16), or O-methyl-L-tyrosine. See FIG. 2.

The identity of the amino acid inserted in response to the TAG codon wasconfirmed by mass analysis of both the intact protein and trypticfragments. The average mass of the intact protein was determined byelectrospray ionization Fourier Transform Ion Cyclotron Resonance MassSpectrometry (FT-ICR MS). The observed value for the monoisotopic massfrom the cluster next to the internal calibrant was 18096.002 Da, whichis within 5 ppm of the theoretical mass of 18095.908 Da and clearlydemonstrates the incorporation of O-methyl-L-tyrosine.

For this experiment a DHFR mutant lacking the C-terminal His tag wasused and purified by methotrexate affinity chromatography. In the mutantprotein, the third codon was changed to TAG, and the fourth codon waschanged from CTG to ATG to improve the amber suppression efficiency,resulting in a Leu4Met mutation.

This result also indicates that other endogenous E. coli synthetases donot utilize O-methyl-L-tyrosine as a substrate. Liquid chromatographytandem mass spectrometry of tryptic digests was carried out to confirmthe sequence of the N-terminal peptide. An example of a tandem MSspectrum is shown in FIG. 3. The doubly charged precursor ion at 691.5Da, corresponding to the N-terminal tryptic peptide MIY*MIAALAVDR (SEQID NO: 77), was selected and fragmented in an ion trap mass spectrometer(ITMS). The fragment ion masses could be unambiguously assigned as shownin FIG. 3, confirming the site-specific incorporation ofO-methyl-L-tyrosine. Neither the protein mass spectra nor the trypticpeptide maps gave any indications of the incorporation of tyrosine orother amino acids in place of O-methyl-L-tyrosine—from thesignal-to-noise ratio of the protein mass spectra a minimum 95%incorporation purity for O-methyl-L-tyrosine was obtained.

Taken together, the cell growth, protein expression and massspectrometry experiments demonstrate that the mtRNA_(CUA) ^(Tyr)/mutantTyrRS (LWJ16) orthogonal pair is capable of selectively insertingO-methyl-L-tyrosine into proteins in response to the amber codon with afidelity rivaling that of the natural amino acids.

Analysis of the sequence of the mutant TyrRS (LWJ16) that charges themtRNA_(CUA) ^(Tyr) with O-methyl-L-tyrosine revealed 12 nucleotidechanges, two of which were silent. The ten nonsilent mutations resultedin the following amino acid residue substitutions relative to wild typeTyrRS: Tyr32, which hydrogen bonds to the aryl oxygen atom of the nativesubstrate tyrosine, was mutated to Gln; Asp158, which hydrogen bonds tothe hydroxyl group of tyrosine, was mutated to Ala; Glu107, whichhydrogen bonds to Asp158, was mutated to Thr; and Leu162, which islocated at the bottom of the binding pocket, was mutated to Pro. Basedon the x-ray crystal structure of the homologous B. stearothermophilusTyrRS, it can be speculated that loss of the hydrogen-bonding networkbetween Tyr32, Asp158 and substrate tyrosine should disfavor binding oftyrosine to the mutant TyrRS (LWJ16). Indeed, mutation of Asp176 (whichcorresponds to Asp158 in M. jannaschii) of B. stearothermophilus TyrRSyields inactive enzyme (G. D. P. Gray, H. W. Duckworth, A. R. Fersht,FEBS 318, 167-71 (1993)). At the same time, the Asp158Ala and Leu162Promutations create a hydrophobic pocket that allows the methyl group ofO-methyl-L-tyrosine to extend further into the substrate-binding cavity.Other important catalytic residues in the active site, which bind to theribose or the phosphate group of the adenylate, were unchanged after tworounds of DNA shuffling. Detailed analysis of these mutations awaits thethree-dimensional structure of the mutant TyrRS (LWJ16).

Kinetics of adenylate formation of O-methyl-L-tyrosine and tyrosine withATP catalyzed by the mutant TyrRS (LWJ16) were analyzed in vitro using apyrophosphate-exchange assay. The mutant TyrRS (LWJ16) gene with sixhistidines at its C-terminus was cloned into plasmid pQE-60 (Qiagen, CA)to afford plasmid pQE-mJYRS. Protein was purified by immobilized metalaffinity chromatography according to manufacture's protocol (Qiagen,CA). Pyrophosphate (PPi) exchange was carried out at 37° C. in areaction mixture containing 100 mM Tris HCl (pH7.5), 10 mM KF, 5 mMMgCl2, 2 mM ATP, 2 mM NaPPi, 0.1 mg/mL bovine serum albumin,approximately 0.01 microCi/mL [32P]NaPPi, and various concentrations oftyrosine or O-methyl-L-tyrosine. Reactions were initiated with theaddition of the purified mutant TyrRS (LWJ16), and aliquots wereperiodically taken and quenched with 0.2 M NaPPi, 7% perchloric acid,and 2% activated charcoal. The charcoal was filtered and washed with 10mM NaPPi (pH2), then measured by scintillation counting to determine the32P levels in charcoal-adsorbed ATP. Values of kcat and Km werecalculated by direct fitting of the Michaelis-Menten equation usingnonlinear regression analysis.

TABLE 1 Kinetic parameters for the mutant TyrRS (LWJ16) toward tyrosineand O-methyl-L-tyrosine measured by pyrophosphate exchange assay. KcatKm kcat/Km Amino acid (10 − 3 s − 1) (μM) (s − 1M − 1)O-methyl-L-tyrosine 14 ± 1  443 ± 93 32 L-tyrosine 1.8 ± 0.2 5833 ± 9020.31

The results of this analysis are shown in Table 1. The Km for tyrosine(5833 μM) is approximately 13 fold higher than that forO-methyl-L-tyrosine, and the kcat for tyrosine (1.8×10−3 s−1) is 8 folddown relative to that for O-methyl-L-tyrosine. Thus the value of kcat/Kmof the mutant TyrRS (LWJ16) for O-methyl-L-tyrosine is about 100 foldhigher than that of tyrosine. The physiological concentration oftyrosine in E. coli is about 80 μM, which is far below Km value (5833μM) of the mutant TyrRS (LWJ16) for tyrosine. Presumably, theconcentration of O-methyl-L-tyrosine in cells is comparable or greaterthan the Km (443 μM).

Example 2 In Vivo Incorporation of L-3-(2-naphthyl)alanine

The site-specific incorporation of a second unnatural amino acid,L-3-(2-naphthyl)-alanine into proteins in E. coli was accomplished. Thisresult shows that this overall scheme is applicable to a host of aminoacids. No synthetase specific for L-3-(2-naphthyl)-alanine were selectedfrom the mutant TyrRS library produced in Example 1, described above.

An amber stop codon and its corresponding orthogonal amber suppressortRNA, mtRNA_(CUA) ^(Tyr) were selected to encode the unnatural aminoacid (Wang, L.; Schultz, P. G. Chem. Biol. 8, 883-890 (2001)). The M.jannaschii tyrosyl-tRNA synthetase (TyrRS) was used as the startingpoint for the generation of an orthogonal synthetase with unnaturalamino acid specificity. This TyrRS does not aminoacylate any endogenousE. coli tRNAs (Steer, B. A.; Schimmel, P. J. Biol. Chem., 274,35601-35606 (1999)), but aminoacylates the mtRNA_(CUA) ^(Tyr) withtyrosine (Wang, L.; Magliery, T. J.; Liu, D. R.; Schultz, P. G. J. Am.Chem. Soc., 122, 5010-5011 (2000)). L-3-(2-naphthyl)-alanine was chosenfor this study since it represents a significant structural perturbationfrom tyrosine and may have novel packing properties.

To change the amino acid specificity of the TyrRS so that it charges themtRNA_(CUA) ^(Tyr) with L-3-(2-naphthyl)-alanine and not any common 20amino acids, a library of M. jannaschii TyrRS mutants was generated andscreened. Based on an analysis of the crystal structure of thehomologous TyrRS from Bacillus stearothermophilus (Brick, P.; Bhat, T.N.; Blow, D. M. J. Mol. Biol., 208, 83-98 (1989)) five residues (Tyr³²,Asp¹⁵⁸, Ile¹⁵⁹, Leu¹⁶², and Ala¹⁶⁷) in the active site of M. jannaschiiTyrRS that are within 7 Å of the para position of the aryl ring oftyrosine were mutated. To reduce the wild-type synthetase contaminationin the following selection, these residues (except Ala¹⁶⁷) were firstall mutated to alanine. The resulting inactive Ala₅ TyrRS gene was usedas a template for polymerase chain reaction (PCR) random mutagenesiswith oligonucleotides bearing random mutations at the correspondingsites.

The mutant TyrRS library was first passed through a positive selectionbased on suppression of an amber stop codon at a nonessential position(Asp112) in the chloramphenicol acetyltransferase (CAT) gene. Cellstransformed with the mutant TyrRS library and the mtRNA_(CUA) ^(Tyr)gene were grown in minimal media containing 1 mML-3-(2-naphthyl)-alanine and 80 μg/mL chloramphenicol. Cells can surviveonly if a mutant TyrRS aminoacylates the mtRNA_(CUA) ^(Tyr) with eithernatural amino acids or L-3-(2-naphthyl)-alanine. The surviving cellswere then grown in the presence of chloramphenicol and the absence ofthe unnatural amino acid. Those cells that did not survive must encode amutant TyrRS that charges the mtRNA_(CUA) ^(Tyr) withL-3-(2-naphthyl)-alanine, and were picked from a replica plate suppliedwith the unnatural amino acid. After three rounds of positive selectionfollowed by a negative screen, four mutant TyrRS's were characterizedusing an in vivo assay based on the suppression of the Asp112TAG codonin the CAT gene. In the absence of L-3-(2-naphthyl)-alanine, cellsexpressing the selected TyrRS and the mtRNA_(CUA) ^(Tyr) survived in 25to 35 μg/mL chloramphenicol on minimal media plates containing 1%glycerol and 0.3 mM leucine (GMML plate); in the presence ofL-3-(2-naphthyl)-alanine, cells survived in 100 to 120 μg/mLchloramphenicol on GMML plates. Compared to the IC₅₀ value in theabsence of any TyrRS (4 μg/mL chloramphenicol), these results indicatethat the selected TyrRS's accept L-3-(2-naphthyl)-alanine, but alsostill charge natural amino acids to some degree.

To further reduce the activity of the mutant TyrRS toward natural aminoacids, one round of DNA shuffling was carried out using the above fourmutant genes as templates. The resulting mutant TyrRS library was passedthrough two additional rounds of positive selections and negativescreens. One mutant TyrRS(SS12-TyrRS) was evolved, whose activity fornatural amino acids was greatly reduced (IC₅₀=9 μg/mL chloramphenicol)while its activity toward L-3-(2-naphthyl)-alanine was enhanced(IC₅₀=150 μg/mL chloramphenicol).

The results of the above described in vivo CAT assays using variousmutant Tyr RS are shown in Table 2. A pYC-J17 plasmid was used toexpress the mtRNA_(CUA) ^(Tyr) gene and the chloramphenicolacetyltransferase gene with an amber stop codon at Asp112. A pBK plasmidwas used to express TyrRS, and was cotransformed with pYC-J17 into E.coli DH10B. Cell survival on GMML plates was titrated in the presence ofdifferent concentrations of chloramphenicol.

TABLE 2 In vivo chloramphenicol acetyltransferase assay of mutant TyrRS.IC₅₀ (μg/mL of chloramphenicol) Mutant TyrRS No L-3-(2-naphthyl)-Ala AddL-3-(2-naphthyl)-Ala no TyrRS 4 4 wt TyrRS 240 240 After selectionS1-TyrRS 30 120 S2-TyrRS 30 120 S3-TyrRS 25 110 S4-TyrRS 35 100 AfterDNA shuffling SS12-TyrRS 9 150

An L-3-(2-naphthyl)-alanine mutant of mouse dihydrofolate reductase(DHFR) was generated and characterized to confirm the ability of themtRNA_(CUA) ^(Tyr)/SS12-TyrRS pair to site-specifically incorporateL-3-(2-naphthyl)-alanine in response to an amber stop codon. The Tyr163codon of the mouse DHFR gene was mutated to TAG, and a His6 tag wasadded to the COOH-terminus of DHFR to facilitate protein purificationusing Ni2+ affinity chromatography. As a positive control, wild-type M.jannaschii TyrRS was coexpressed with the mtRNA_(CUA) ^(Tyr) resultingin efficient suppression of the TAG codon with tyrosine (FIG. 4). WhenSS12-TyrRS was coexpressed with the mu tRNA_(CUA) ^(Tyr) in the presenceof 1 mM L-3-(2-naphthyl)-alanine, full-length mouse DHFR was alsogenerated (with yield of 2.2 mg/L in liquid GMML minimal medium). In theabsence of either L-3-(2-naphthyl)-alanine, mtRNA_(CUA) ^(Tyr), orSS12-TyrRS, no full length DHFR was produced. A penta-His antibody wasused to detect the His6 tag at the COOH-terminus of DHFR in a Westernblot. No DHFR could be detected in the absence of each of the abovethree components.

Tryptic digests of the L-3-(2-naphthyl)-alanine mutant of mouse DHFRwere analyzed by MALDI FT-ICR and liquid chromatography tandem massspectrometry to confirm unambiguously the incorporation ofL-3-(2-naphthyl)-alanine. The peptide map of the internally calibrateddigest shows a major peak at 1867.962, which is within 3.5 ppm of thetheoretical mass of the tryptic peptide LLPEX*TGVLSEVQEEK (SEQ ID NO:78)where X* represents the L-3-(2-naphthyl)-alanine residue (Pro164 wasmutated to Thr to improve the amber suppression efficiency). Further,the interpreted tandem mass spectrum of precursor ion at m/z 934.5,which corresponds to the doubly charged ion of the peptide of interestis shown in FIG. 5. The sequence information gleaned from the spectrumclearly demonstrates the site-specific incorporation ofL-3-(2-naphthyl)-alanine into the protein. Neither peptide maps nor LCMS/MS runs produced any indication of mutants in which theL-3-(2-naphthyl)-alanine residue is substituted by other amino acids.The signal-to-noise ratio of more than 1500 observed in the peptide mapsshows a fidelity in the incorporation of L-3-(2-naphthyl)-alanine ofbetter than 99.8%.

The evolved SS12-TyrRS has the following mutations: Tyr32→Leu32,Asp158→Pro158, Ile159→Ala159, Leu162→Gln162, and Ala167→Val167.Corresponding residues from B. stearothermophilus are Tyr³² (Tyr³⁴),Asp¹⁵⁸ (Asp¹⁷⁶), Ile¹⁵⁹ (Phe¹⁷⁷), Leu¹⁶² (Leu¹⁸⁰), and Ala¹⁶⁷ (Gln¹⁸⁹)with B. stearothermophilus TyrRS residues in parenthesis.

Based on the crystal structure of the homologous B. stearothermophilusTyrRS, the mutations of Tyr32→Leu32 and Asp158→Pro158 probably result inthe loss of hydrogen bonds between Tyr32, Asp158, and the nativesubstrate tyrosine, thus disfavoring the binding of tyrosine toSS12-TyrRS. Most residues are mutated to amino acids with hydrophobicside chains, which are expected to favor binding ofL-3-(2-naphthyl)-alanine.

In summary, the cell growth, protein expression, and mass spectrometryexperiments demonstrate that the mtRNA_(CUA) ^(Tyr)/SS12-TyrRS pair iscapable of selectively inserting L-3-(2-naphthyl)-alanine into proteinsin response to the amber codon with fidelity rivaling that of thenatural amino acids.

Example 3 In Vivo Incorporation of Amino-, Isopropyl-, orAllyl-Containing Tyrosine Analogues

A FACs based screening system was used to rapidly evolve three highlyselective synthetase variants that accept amino-, isopropyl-, orallyl-containing tyrosine analogues. The system included a multipurposereporter plasmid used for application of both positive and negativeselection pressure and for the facile and quantitative evaluation ofsynthetase activity. A chloramphenicol acetyl transferase (CAT) markerallowed positive selection for activity of the M. jannaschiityrosyl-tRNA synthetase (TyrRS). A T7 polymerase/GFP reporter systemallowed assessment of synthetase activity within cells grown in both thepresence and absence of an unnatural amino acid. Fluorescence activatedcell sorting (FACS) was used to screen against synthetase variants thataccept natural amino acids, while visual and fluorimetric analyses wereto assess synthetase activity qualitatively and quantitatively,respectively.

Design of an amplifiable fluorescence reporter system. Efforts todevelop a versatile screening system for the assessment of synthetaseactivity in living cells initially arose out of a desire for a greaterdegree of control over the selective pressure applied to populations ofsynthetase variants, especially negative selective pressure. As thesystem was to be used to assess the activities of large numbers ofsynthetase variants, a reporter was sought that would be amenable tohigh-throughput screening. In addition, a reporter that would allow forfacile qualitative and quantitative evaluation of synthetase activitywas desired. To meet these requirements, a fluorescence-based screen wasdesigned. The system was based on the synthetase-dependent production ofGFPuv, a variant of the green fluorescent protein that has beenoptimized for expression in E. coli (Crameri, A., Whitehorn, E. A.,Tate, E. & Stemmer, W. P., Nature Biotechnol. 1996, 14, 315-319). Thisfluorophore is amenable to use in FACS and fluorimetry, as well asvisual inspection on plates and in liquid culture. The system wasdesigned such that synthetase-dependent suppression of selector, e.g.,amber nonsense codons would result in the production of a fluorescencesignal. In order to maximize the sensitivity of the reporter, it wasmade amplifiable by placement of the amber codons within the gene for T7RNA polymerase, which was designed to drive expression of the GFPuvreporter gene in analogy to other amplifiable intracellular reportersystems (Lorincz, M., Roederer, M., Diwu, Z., Herzenberg, L. A., Nolan,G. P. Cytometry, 1996, 24, 321-329; Zlokarnik, G., Negulescu, P. A.,Knapp, T. E., Mere, L., Burres, N., Feng, L., Whitney, M., Roemer, K. &Tsien, R. Y., Science, 1998, 279, 84-88). The T7 RNA polymerase gene wasplaced under control of the arabinose promoter in order to allow facileoptimization of the production of the RNA transcript for ambercodon-containing T7 RNA polymerase.

Optimization of the T7 RNA polymerase/GFPuv reporter system. Amedium-copy reporter plasmid, pREP, was designed to expressamber-containing T7 RNA polymerase variants under control of thearabinose promoter and the GFPuv gene under control of the T7 promoter(FIG. 6 a). A series of twelve T7 RNA polymerase variants, designed tooptimize synthetase-dependent fluorescence enhancement (FIG. 6 b), wereinserted into pREP to create plasmids pREP(1-12). All variants containedan N-terminal leader sequence of seven amino acids (MTMITVH, SEQ IDNO:79) and 1-3 amber stop codons (TAG). Variants 1-3 contained one, two,and three amber stop codons, respectively, substituted for the originalmethionine at position one (M1), just downstream of the leader sequence.Variants 49 contained an amber codon substituted for D10, R96, Q107,A159, Q169, or Q232, respectively, which were predicted to be located inloop regions of the structure (Jeruzalmi, D. & Steitz, T. A., EMBO J.,1998, 17, 4101-4113). Variants 10-12 contained amber stop codonssubstituted at postions M1 and either Q107, A159, or Q232, respectively.Plasmid constructs were evaluated by fluorimetry and flow cytometry oflive cells for fluorescence enhancement using a compatible plasmidcontaining the orthogonal glutaminyl-tRNA synthetase and GlutaminetRNA_(CUA) from S. cerevisiae. Plasmids pREP(1-12) were found to providevarying levels of synthetase-dependent fluorescence enhancement, withthe best construct, pREP(10) exhibiting 220-fold greater fluorescence byfluorimetry (FIG. 6 c) and ˜400-fold greater median fluorescence bycytometry (FIG. 6 d) in cells containing the wild type synthetase versusan inactive mutant. Substitution of a variety of functional groups atpositions corresponding to the amber codons within pREP(10) demonstratethat position 107 within T7 RNA polymerase is highly permissive.

Construction of a multipurpose reporter plasmid. In order to construct amultipurpose plasmid to be used both for selecting and screeningvariants of a M. jannaschii TyrRS, plasmid pREP(10) was combined withplasmid pYC-J17 (Wang, L, Brock, A., Herberich, B. & Schultz, P. G.,Science, 2001, 292, 498-500) to obtain pREP/YC-JYCUA (FIG. 7 b). PlasmidpREP/YC-JYCUA was assayed for function with a compatible plasmidexpressing a variant of M. jannaschii TyrRS (pBK-mJYRS; Wang, L, Brock,A., Herberich, B. & Schultz, P. G., Science, 2001, 292, 498-500)selective for incorporating O-Methyl-Tyrosine (OMY). Cells containingpREP/YC-JYCUA and pBK-mJYRS, grown in the presence of OMY, exhibited achloramphenicol (Cm) IC₅₀ value of 120 micrograms/ml, identical to thatobtained using plasmid pYC-J17, and a fluorescence enhancement of330-fold for cells grown in the presence versus the absence of OMY, asmeasured by fluorimetry.

Evolution of the substrate specificity of the M. jannaschii tyrosyl-tRNAsynthetase. Results have shown that the amino acid side chain bindingpocket of the M. jannaschii TyrRS can be evolved to selectivelyaccommodate chemical groups other than the phenol side chain of tyrosine(Wang, L, Brock, A., Herberich, B. & Schultz, P. G., Science, 2001, 292,498-500; Wang, L., Brock, A. & Schultz, P. G. J. Am. Chem. Soc. 2002,124, 1836-1837). We sought to further explore the generality ofunnatural amino acid accommodation by M. jannaschii TyrRS by challengingthe enzyme to accept four new functionalities: p-Isopropyl-Phenylalanine(pIF), p-Amino-Phenylalanine (pAF), p-Carboxyl-Phenylalanine (pCF), orO-Allyl-Tyrosine (OAT) (FIG. 7 b). A library of M. jannaschii TyrRSvariants containing randomizations at positions Y32, E107, D158, I159,and L162 (Wang, L, Brock, A., Herberich, B. & Schultz, P. G., Science,2001, 292, 498-500), residues thought to form the binding pocket for thepara position of the tyrosyl ring, was introduced into cells containingplasmid pREP/YC-JYCUA. These cells, encompassing a library diversity of˜10⁹, were used to begin four evolution experiments to identifysynthetase variants selective for pIF, pAF, pCF, or OAT (FIG. 7 c). Twocycles of positive selection were carried out by allowing the cellcultures to grow to saturation in the presence of Cm and one of the fourunnatural amino acids. Cell aliquots were removed following the secondcycle of positive selection and used to inoculate a new culturecontaining no added amino acid or Cm, and the culture was again allowedto grow to saturation. At this point, cells that fluoresce are likely tocontain synthetase variants that can accept one of the 20 natural aminoacids. Approximately 10⁸ cells from each line were subjected to negativescreening using FACS in order to eliminate natural amino acid-acceptingsynthetase variants. The non-fluorescent cells were collected andamplified through growth to saturation. These amplified cells were usedto inoculate a new culture for a final cycle of positive selection inliquid culture containing unnatural amino acid and Cm. Following growthto saturation, each population of cells was plated on media containing0, 30, 60, or 100 micrograms/mL Cm and either 0 or 1 mM of theappropriate unnatural amino acid.

Identification and characterization of evolved synthetase variants. Cmplates supplemented with pIF, pAF, and OAT produced 10-100-fold greaternumbers of fluorescent colonies than plates containing no added aminoacid. In contrast, plates for the pCF population produced the samenumber of fluorescent colonies with or without addition of pCF. The tenlargest fluorescent colonies were picked for each of the pIF, pAF, andOAT populations from unnatural amino acid-containing plates and grown tosaturation in liquid media with or without added unnatural amino acid. Aqualitative assessment of fluorescence production was made visually withthe use of a hand-held long-wavelength ultraviolet lamp (FIG. 8 a).

Synthetase variants corresponding to clones producing significantdifferences in fluorescence were sequenced. All ten clones from the pIFand pAF populations had identical sequences, while three differentclones were identified from the OAT population. Amino acid changesoccurred within the five randomized sites in all clones, with theexception of two additional substitutions within the pIF-tRNA synthetase(pIF-RS) variant. The activities of the different clones werequantitatively assessed. Fluorescence was measured fluorimetrically forcells grown in liquid culture in the presence or absence of unnaturalamino acid (FIG. 8 b). The Cm IC₅₀s were determined by plating the cellson varying concentrations of Cm in the presence or absence of unnaturalamino acid (FIG. 8 c).

A myoglobin gene containing an amber codon in the fourth position wasused to assess the production of unnatural amino acid-containingprotein. The gene was expressed in cells, using the pIF-RS, pAF-RS, orOMY-RS variant, respectively, in either the presence or absence of pIF,pAF, or OAT (FIG. 8 d). Protein yields were comparable for all threevariants, ranging from 1-2 milligrams of protein per liter of unnaturalamino acid-containing cell culture. In contrast, protein production wasvirtually undetectable in cultures grown in the absence of unnaturalamino acid. Proteins were analyzed by electrospray mass spectrometry,giving masses of 18457.40±0.81 (18457.28 expected) for thepIF-containing protein, 18430.30±0.27 (18430.21 expected) for thepAF-containing protein. Activity measurements obtained using the CmIC₅₀, fluorimetry, and protein expression analyses correlated well,however the activity of the pIF-RS appears to be somewhat underestimatedby fluorimetry. As compared to other assays, the disproportionately lowfluorimetry measurement for the pIF-RS variant, shows that T7 RNApolymerase may be partially destabilized upon incorporation of the pIFanalogue, despite the apparent permissivity of the amber positionswithin the reporter.

Utility of the multipurpose reporter system. The reporter systemdescribed here allows the use of a single multipurpose plasmid for bothpositive selection and negative screening, obviating the need to shuttleplasmids between alternating rounds of positive and negative selection.A total of only three rounds of positive selection and one round ofnegative screening were required to enable the identification ofsynthetase variants that selectively accept desired unnatural aminoacids. These features allow evolution experiments to be carried out in amatter of days. The screening system can be used to readily identifyactive synthetase variants using agar plates containing unnatural aminoacid and to individually assay the amino acid specificity of thevariants.

As described above, the T7 RNA polymerase/GFP system can be used toquantitatively compare the activities of synthetase variants. Theavailability of the three OAT-RS clones described here and a differentOAT-RS clone derived independently from the same library using apositive/negative selection based on CAT and barnase (Table 2) allowsthe possibility of comparing the two different evolution systems interms of the synthetase variants resulting from each (FIG. 9). Thisanalysis reveals that the three clones derived from positive selectionand negative screening exhibit slightly lower levels of fluorescence inthe presence of OAT, but ˜10-fold lower background levels in the absenceof the unnatural amino acid. The fluorescence enhancement for cellsgrown in the presence versus the absence of the unnatural amino acid isthus about 6-fold higher for cells expressing OAT-RS (1) from selectionand screening than for cells expressing the OAT-RS clone derived frompositive/negative selection using barnase. Although it is not clearwhether this example is representative, these data suggest that the T7RNA polymerase/GFP system may allow more stringency in selecting againstsynthetase variants that are promiscuous towards natural amino acidsubstrates. However, the fluorescence enhancement for cells grown in thepresence versus the absence of an unnatural amino acid is expected torepresent a lower limit for the fidelity of unnatural amino acidincorporation, as competition of unnatural amino acids for being boundby an evolved synthetase variant would reduce binding of natural aminoacids. Moreover, although high fidelity is clearly desirable, there islikely to be a trade-off between fidelity and overall synthetaseactivity, which may depend on the desired application.

Generality of aminoacyl tRNA synthetase evolution. Previous results andthose presented here demonstrate that the amino acid side chain bindingpocket of the M. jannaschii TyrRS is quite malleable. The enzyme can beevolved to accommodate a variety of functionalities in place of thephenol side chain of tyrosine and can do so with high selectivity. Inthis application it was demonstrated that enzyme can be evolved toaccommodate an amine, isopropyl, or allyl ether functionality at thepara position of the tyrosine ring, instead of hydroxyl.

Plasmid Construction. Plasmid pREP (FIG. 6 a) was constructed byinsertion of a BamHI/ApaLI overlap PCR fragment containing the T7 RNApolymerase gene upstream of an rrnB transcription termination region,followed by an ApaLI/AhdI overlap PCR fragment containing the araC geneand ara promoter region from the pBAD/Myc-His A plasmid (Invitrogen; fortranscriptional control of the T7 RNA polymerase gene) and the GFPuvgene (Clontech; upstream of the T7 terminator region and downstream ofthe T7 promoter) between the AhdI/BamHI sites of plasmid pACYC177 (NewEngland Biolabs). Plasmids pREP(1-12) were constructed by replacement ofan HpaI/ApaLI fragment of T7 RNA polymerase with overlap PCR fragmentscontaining amber mutations at the positions described. PlasmidpREP/YC-JYCUA was constructed by ligation of an Afel/SacII fragment frompREP(10) and an EarI(blunted)/SacII fragment from pYC-J17 (Wang, L,Brock, A., Herberich, B. & Schultz, P. G., Science, 2001, 292, 498-500).The desired construct was identified following transformation into cellscontaining plasmid pQ screening for fluorescence.

Plasmid pQ was constructed by triple ligation of a AatII/SalI overlapPCR fragment containing the SCQRS downstream of the lac promoter regionand upstream of the E. coli QRS termination region, a SalI/AvaI overlapPCR fragment containing the S. cerevisiae tRNA(CUA)^(Gln) downstream ofthe lpp promoter region and upstream of an rrnC termination region, andthe AvaI/AatII fragment of pBR322 (New England Biolabs). Plasmid pQD wasconstructed by replacement of pQ fragment between BamHI and BglII with aBamHI/BglII fragment of the SCQRS (D291A) mutant.

Plasmid pBAD/JYAMB-4TAG was constructed by insertion of a PCR fragmentof the S4Amber mutant of myoglobin, containing a C-terminal 6His-tag,into the pBAD/YC-JYCUA plasmid, a hybrid of plasmid pYC-J17 (Wang, L,Brock, A., Herberich, B. & Schultz, P. G., Science, 2001, 292, 498-500)and pBAD/Myc-His A (Invitrogen) containing the gene for MjYtRNA_(CUA),and the pBAD promoter and cloning regions for heterologous expression ofan inserted gene.

Fluorimetric and cytometric analyses. Single colonies containing desiredplasmids were used to inoculate 2-mL GMML cultures containing theappropriate antibiotics, 0.002% Arabinose, and an appropriate unnaturalamino acid, if desired. Cultures were grown to saturation and cells (200μL) were pelleted and resuspended in 1 mL phosphate-buffered saline(PBS). Cell concentrations were analyzed by absorbance at 600 nm andfluorescence levels were measured at 505 nm with excitation at 396 nmusing a FluoroMax-2 fluorimeter. Cells suspended in PBS were analyzedcytometrically. To evaluate the permissivity of the amber positionswithin the T7 polymerase gene of pREP(10), the reporter plasmid wastransformed into a panel of suppressor strains, which were subsequentlyanalyzed fluorimetrically.

Evolution of aminoacyl-tRNA synthetase variants. M. jannaschii TyrRSvariants randomized at positions Y32, E107, D158, I159, and L162 (Wang,L, Brock, A., Herberich, B. & Schultz, P. G., Science, 2001, 292,498-500) were transformed into DH10B E. coli cells (Life Technologies)containing pREP/YC-JYCUA to generate a library with a diversity of ˜10⁹.Transformants were allowed to recover in SOC medium for 60 min at 37°C., and were grown to saturation in LB medium. To begin an initialpositive selection, 2 mL of library culture, pelleted and resuspended inGMML medium, was used to inoculate 500 mL of GMML containing 25 μg/mLTetracycline (Tet), 35 μg/mL Kanamycin (Kn), and 1 mM pIF, pAF, pCF, orOAY. After incubation for 3 hr at 37° C., Cm was added to a finalconcentration of 75 μg/mL and cells were grown to saturation (˜48 hr).For the second positive selection, a 100-mL GMML culture containing Tet,Kn, 75 μg/mL Cm, and 1 mM pIF, pAF, pCF, or OAY was inoculated withcells from the initial positive selection (500 μL) and grown tosaturation at 37° C. (˜24-36 hr). In preparation for negative screening,a 25-mL GMML culture containing Tet, Kn, and 0.02% arabinose (Ara) wasinoculated with cells from the second positive selection (100 μL,pelleted and resuspended in GMML) and grown to saturation at 37° C. (˜24hr). Ara-induced cells grown in the absence of unnatural amino acids (1mL) were pelleted and resuspended in 3 mL of phosphate-buffered saline(PBS). Cells were sorted for lack of expression of GFPuv using a BDISFACVantage TSO cell sorter with a Coherent Enterprise II ion laser withexcitation at 351 nm and emissions detected using a 575/25 nm bandpassfilter. Collected cells were diluted in at least 10 volumes of LB,containing Tet and Kn, and grown to saturation. To begin the third roundof positive selection, 100 μL of cells from the negative screen werepelleted, resuspended in GMML, and used to inoculate 25 mL of GMMLcontaining Tet, Kn, and 1 mM pIF, pAF, pCF, or OAY. After incubation for3 hr at 37° C., Cm was added to a final concentration of 75 μg/mL andcells were grown to saturation (˜24 hr). Following the third positiveselection, cells were plated on GMML/agar containing Tet, Kn, 0.002%Ara, 0, 75, or 100 μg/mL Cm, and 0 or 1 mM pEF, pAF, pCF, or OAY, andgrown for 48 hr at 37° C.

-   Expression and characterization of unnatural amino acid-containing    proteins. DH10B cells cotransformed with pBAD/JYAMB-4TAG and the    appropriate pBK plasmid were used to inoculate a 100-mL GMML starter    culture containing Kn and Tet, which was grown to saturation. A    500-mL culture containing Kn, Tet, 0.002% Ara, 5 μM FeCl₃, and the    desired unnatural amino acid (or none) was inoculated with 50 mL of    the starter culture and grown to saturation (˜18 hr). Cultures were    pelleted, sonicated, and the myoglobin protein isolated according to    the protocol of the QiaExpressionist (Qiagen) His-tag purification    kit. Proteins were analyzed electrophoretically on a 12-20% gradient    SDS polyacrylamide gel and by electrospray mass spectrometry.

Example 4 Creation of an Autonomous 21 Amino Acid Bacterium

As described above, the common twenty amino acids are conserved acrossall known organisms. However, an expanded genetic code is providedherein, e.g., for added functionality, structure determination and thelike. To determine whether the expanded genetic code is advantageous toa cell, e.g., with a particular unnatural amino acid, an autonomousbacterium that produces and incorporates the unnatural amino acid ofinterest is desirable. The present invention provides such an autonomoustwenty-one amino acid organism, and the results can be extended to theproduction of additional amino acid organisms, e.g., 22 amino acidorganisms and the like. To produce an autonomous bacterium, threefactors are typically considered: (i) the ability to synthesize a newamino acid from simple carbon sources; (ii) an aminoacyl synthetase thatuniquely utilizes this new amino acid and no other; and (iii) a tRNAthat is acylated by that synthetase and no other, and which delivers theamino acid into proteins in response to a codon that does not encode anyother amino acid.

A great deal of effort has been made toward in vivo incorporation of newamino acids to the genetic code but most of these do not have theincorporation specificity to generate a healthy 21 amino acid bacterium.See, e.g., Hest, J. C. M.v., K. L. Kiick, and D. A. Tirrell, J. Am.Chem. Soc., 2000. 122: p. 1282; Hamano-Takaku, F., et al., J. Biol.Chem., 2000. 275: p. 40324; and Budisa, N., et al., FASEB J., 1999. 13:p. 41-51. However, it has recently been shown that that one could addnew components to the translational machinery of E. coli andsite-specifically incorporate a variety of new amino acids into proteinsin vivo, e.g., with high fidelity. See, e.g., Wang, L., et al., Science,2001, 292: p. 498-500 and Wang, L. and P. G. Schultz, Chem. Comm., 2002:p. 1-10. See, also, co-filed patent application “Methods andCompositions for the Production of Orthogonal tRNA-tRNA SynthetasePairs,” by Schultz et al., U.S. patent application Ser. No. 10/126,931(Attorney Docket Number 54-000130), filed Apr. 19, 2002.

The present invention combines the above technology with a biosyntheticpathway system to produce an autonomous twenty-one amino acid bacterium.In addition, the present invention addresses the question of whethersuch organisms have or can be evolved to have an evolutionary advantageover organisms that use the twenty natural amino acids.

A completely autonomous bacterium typically comprises a biosyntheticpathway system, e.g., for producing an unnatural amino acid, and atranslation system for incorporating the unnatural amino acid into oneor more proteins in the bacterium. The translation system typicallycomprises an aminoacyl synthetase that uniquely utilizes this unnaturalamino acid and no other, and a tRNA that is acylated by that synthetaseand no other, and which delivers the unnatural amino acid into proteinsin response to a codon that does not encode any other amino acid. In oneembodiment, the biosynthetic pathway system genes, aminoacyl synthetasegenes, and tRNA genes are typically positioned on separate plasmids tomaximize control of the modified bacteria.

In one example, the unnatural amino acid, p-aminophenylalanine (pAF), isbiosynthetically produced and incorporated into proteins in vivo. pAF isoptionally selected as a unnatural amino acid for an autonomous cell,e.g., based on its interesting physical properties, e.g., a donatingeffects, hydrogen bonding properties, and weak basicity, its lack oftoxicity to E. coli, and the fact that it is a known secondarymetabolite. Moreover, the genes that lead to the production of pAF as ametabolic intermediate in the production of chloramphenicol andpristinamycin have been identified in Streptomyces Venezuelae andStreptomyces pristinaespiralis, respectively. See, e.g., Yanai, K. ande. al., Streptomyces venezuelae genes papA, papB, papC, in PCT Int.Appl. 2001, Meiji Seika Kaisha Ltd.: Japan. p. 1-83; and Blanc, V., etal., Identification and analysis of genes from Streptomycespristinaespiralis encoding enzymes involved in the biosynthesis of the4-dimethylamino-L-phenylalanine precursor of pristinamycin I. MolecularMicrobiology, 1997. 23(2): p. 191-202. As discussed above, pAF isoptionally synthesized in E. coli from chorismate 2 (a biosyntheticintermediate in the synthesis of aromatic amino acids) using the S.Venezuelae enzymes PapA, PapB, and PapC together with an E. coliaminotrasferase. A plasmid, e.g., as provided in FIG. 15A is optionallyused to transform a cell to provide a cell that synthesizes its ownsupply of pAF in vivo. An example plasmid for use in the biosynthesis ofpAF in vivo is provided by SEQ. ID. NO.:67. SEQ ID NO.:68 provides thesequences for the individual genes papABC that encode the enzymes thatare used to carry out the conversion of chorismate to pAF.

Once a cell is modified to produce an unnatural amino acid, e.g., pAF,O-methyl-L-tyrosine, a glycoslyated amino acids, L-dopa or the like, thecell is also typically modified by the addition of a translation systemfor incorporating the unnatural amino acid into one or more proteinswithin the cell. The translation system is typically provided to thecell via a separate plasmid than that by which the cell is modified tocontain the biosynthetic pathway system as this allows closer controlover the functions of the plasmids in the cell, e.g., regarding thenumber of copies, promoters, etc.

The translation machinery typically comprises an orthogonal tRNA/RSpair, e.g., as provided by co-filed patent application “Methods andCompositions for the Production of Orthogonal tRNA-tRNA SynthetasePairs,” by Schultz et al., U.S. patent application Ser. No. 10/126,931(Attorney Docket Number 54-000130), filed Apr. 19, 2002. For example, anorthogonal tRNA/RS pair for pAF is optionally progenerated using aMethanococcus jannaschii tyrosyl-tRNA synthetase (TyrRS) and mutanttyrosine amber suppressor tRNA (mtRNA_(CUA) ^(Tyr)) pair as a startingpoint. See, e.g., Wang, L., et al., A new functional suppressortRNA/aminoacyl-tRNA synthetase pair for the in vivo incorporation ofunnatural amino acids into proteins. J. Am. Chem. Soc., 2000 122: p.5010-5011; and Wang, L. and P. G. Schultz, Chem. and Biol., 2001, 8:883.

For example, a pAF specific synthetase (pAFRS) is optionally generatedby modifying the amino acid specificity of the M. jannaschii TyrRS toaccept pAF and not any of the common twenty amino acids. See, e.g.,Wang, L., et al., Expanding the genetic code of Escherichia coli.Science, 2001, 292: p. 498-500; Wang, L. and P. G. Schultz, Expandingthe Genetic Code. Chem. Comm., 2002: 1:1-10; and Wang, L., A. Brock, andP. G. Schultz, Adding L-3-(2-naphthyl)alanine to the genetic code of E.coli. J. Am. Chem. Soc., 2002. 124: p. 1836. A combination of positiveselections and negative screens are optionally used to identify a pAFrsenzyme from a library of TyrRS variants containing random amino acids atfive positions, e.g., Tyr32, Glu107, Asp158, Ile159, and Leu162. Asingle reporter plasmid is optionally used for both selection andscreening, e.g., as described in co-filed patent application “Methodsand Compositions for the Production of Orthogonal tRNA-tRNA SynthetasePairs,” by Schultz et al., U.S. patent application Ser. No. 10/126,931(Attorney Docket Number 54-000130), filed Apr. 19, 2002. The positiveselection is typically based on suppression of a TAG codon at apermissive position within the chloramphenicol acetyltransferase (CAT)gene. (see, e.g., Wang, L., et al., Expanding the genetic code ofEscherichia coli. Science, 2001, 292: p. 498-500 and Pasternak, M., T.J. Magliery, and P. G. Schultz, A new orthogonal suppressortRNA/aminoacyl-tRNA synthetase pair for evolving an organism with anexpanded genetic code. Helvetica Chemica Acta, 2000 83: p. 2277), e.g.,by either pAFor an endogenous amino acid. Cells containing the TyrRSlibrary and reporter plasmid grown in liquid culture containing pAF aretypically selected for survival, e.g., in the presence ofchloramphenicol (Cm). The negative screen based on suppression of twoUAG stop codons at permissive positions within the T7 RNA polymerasegene drives the expression of green fluorescent protein (GFP).Positively selected cells grown in the absence of pAF and Cm, are thentypically screened, e.g., using fluorescence activated cell sorting(FACS) for the lack of fluorescence.

Evolution of pAFrs: The reporter plasmid, pREP(2)/YC-JYCUA, contains thegenes for CAT, T7 RNA polymerase, GFP, and mtRNA_(CUA) ^(Tyr), and aselectable marker for Tet resistance (Santoro unpublished results). TheCAT gene contains a TAG codon substitution at position D112. The T7 RNApolymerase gene contains a seven-amino acid N-terminal leader peptideand TAG substitutions at M1 and Q107. For the positive selection, cellswere grown in GMML minimal media containing 35 μg/ml Kn, 25 μg/ml Tet,75 μg/ml Cm, and 1 mM pAF (Sigma). For the negative screen, cells weregrown in GMML media containing 35 μg/ml Kn, 25 μg/ml Tet, and 0.002%arabinose. FACS was carried out using a BDIS FACVantage TSO cell sorterwith a Coherent Enterprise II ion laser. The excitation wavelength was351 nm and emission was detected using a 575/25 nm bandpass filter.Collected cells were diluted into at least 10 volumes of LB, containingTet and Kn, and grown to saturation.

Addition of pAF biosynthetic pathway: The papA, papB, and papC geneswere PCR amplified from S. Venezuele (ATCC 10712) genomic DNA. Genes,papABC were assembled by overlap PCR and inserted into a pSC101 derivedplasmid, pLASC, and maintained by ampicillin selection. Ribosome bindingsites (rbs) were from the 5′ UTR of LacZ, malE, and cro and placed priorto papA, papB, and papC, respectively. The papABC genes were placedunder control of lac and lpp promotor to afford two pathway plasmidspLASC-lacPW and pLASC-lppPW.

Testing pAF biosynthesis with pAFRS: E. coli DH10B cells harboring threeplasmids, the reporter plasmid (pREP(2)/YC-JYCUA), the synthetase(pAFRS), and the pathway plasmid (pLASC-lacPW or pLASC-lppPW) were grownto saturation in GMML minimal media (pLASC was used for background, nopAF, and 1 mM exogenous pAF trials). DH10B was grown with no plasmids todetermine the background suppression level of the reporter plasmid. Asample of each cell growth was diluted to an OD of 1.0 (600 nm) withwater and 200 μL was pelleted. Cell were suspended in 1 mL 1% PBS andanalyzed using a Fluoromax-2 fluorescent detector (excitation wavelengthwas 351 nm and a peak emission at 505 nm was monitored). DH10B produced1.0×10 4 fluorescent units, while background fluorescence (no pAF added)from the reporter system produced 2.5×10 4 fluorescent units. The lacPW,lppPW, and 1 mM exogenously added pAF produced 7.9×10 4, 3.0×10 6, and3.0×10 4 fluorescent units, respectively. Induction of the lacPW withIPTG was not feasible due its inhibitory affect on the arabinosepromotor in the reporter plasmid, (pREP(2)/YC-JYCUA).

Aromatic amino acid concentration: E. coli DH10B cells harboring thepLASC plasmid and pLASC-lacPW or pLASC-lppPW were grown in GMML minimalmedia (1% glycerol, 0.3 mM leucine) containing 110 μg/ml ampicillin tosaturation. Cells grown with exogenously added pAF contained 1 mM aminoacid at the start of the growth. Cells were harvested by centrifugation(100 ml), washed, 1 ml of water and 0.2 ml of toluene was added. Cellswere shaken at 37° C. 11 for 30 minutes and then separated bycentrifugation. The aqueous layer was filtered (microcon YM-10) andanalyzed by HPLC-MS (Agilent 1100): 5-15 μL of the aqueous layerseparated on Zorbax SB-C18 column (5 μm, 4.6×150 mm) with a gradient ofwater 1% TFA/acetonitrile 1% TFA (95:5) to (5:95) over 10 minutes. Aminoacids were identified by abstracting their MW(+1) from the total ionmass spectrum. The area of the abstracted ion was used to calculateamount of amino acids present in each sample. Cellular concentrationswere based on the amount of water in the cell pellet, 70% by mass.

Expression of protein containing pAF: Plasmid pBAD/JYAMB-4TAG withtetracycline resistance was used to express the Tyr CUA mutRNA geneunder the control of the lpp promotor and rrnC terminator, and themyoglobin gene (with an amber stop codon at Ser4) under the control ofthe arabinose promotor and rrnB terminator. A his6-tag was added to thecarboxy terminus of myoglobin. The TyrRS and pAFRS genes were expressedunder the control of the E. coli GlnRS promotor and terminator on apBR322 derivatived plasmid with kanamycin resistance. The papABC geneswere expressed from pLASC-lacPW or pLASC-lppPW (13) under the control ofthe native terminator. E. coli DH10B cells harboring plasmidpBAD/JYAMB-4TAG, pBK-TyrRS or pBK-pAFRS, and a pLASC derived plasmid(pLASC, pLASC-lacPW or pLASC-lppPW as indicated) were grown in 0.5 L ofminimal media containing 0.002% arabinose. Expression trials withexogenous pAF contained a final concentration of 1 mM pAF (Sigma). Forall trials, cells were grown to saturation (20-30 hrs) in parallel at37° C., pelleted, and protein was purified by Ni +2 affinitychromatography according to manufacturer's protocol under nativeconditions (Qiagen, Valencia, Calif.). Fifteen μl of final proteinsolution (3.5 ml) from each preparation were separated on a 12% SDSpolyacrylamide gel and silver-stained.

Example 5 In Vivo Incorporation of O-methyl-L-tyrosine in an E. coliCell which has been Genetically Engineered to Biosynthesize theUnnatural Amino Acid

As discussed herein, one aspect of the invention is biosyntheticpathways for unnatural amino acids in E. coli. This is accomplished bye.g., addition to the cell of genes for new enzymes or modification ofexisting E. coli pathways. In this example, E. coli was geneticallyengineered to produce the unnatural amino acid O-methyl-L-tyrosine.

Plant O-methyltransferases are enzymes involved in secondary metabolism,which converts a hydroxyl group into a methoxyl group. Two enzymes,(iso)eugenol O-methyltransferase (IEMT) and caffeic acidO-methyltransferase (COMT) (Clarkia brewery) were selected forincorporation into E. Coli. IEMT methylates eugenol/isoeugenol, and COMTmethylates caffeic acid. The substrates of these two enzymes are similarto tyrosine. However, both enzymes have high substrate specificity andmethylation regiospecificity.

A combinatorial approach was used to evolve the substrate specificity ofboth enzymes to tyrosine, thereby converting tyrosine toO-methyl-L-tyrosine. Active sites of the proteins were mutated toproduce large mutant libraries and several rounds of selection werecompleted. Three clones were identified. The clones are characterizedand at least one is selected to generate an E. coli strain thatbiosynthesizes O-methyl-L-tyrosine. This strain of E. coli isgenetically engineered to also express the orthogonal tRNA/RS pairdescribed in Example 1 above, thereby providing a cell for autonomous invivo incorporation of an unnatural amino acid.

Example 6 In Vivo Incorporation of Heavy Atom Amino Acids

Structure-guided drug discovery has historically been a slow, laboriousprocess used in only a modest fraction of drug discovery programs in theindustry. One bottleneck is the phase problem encountered when usingX-ray crystallography to solve protein structure. Typically, the proteinhas to be expressed again in the presence of selenomethionine, whichdoubles the work load and may not necessary result in successfulcrystallization. An alternative method is to soak the crystal in aheavy-atom-containing solution, which may result in crystal crush. Invivo incorporation of heavy-atom containing unnatural amino acids intoproteins is a useful tool to accelerate the solving of protein crystalstructures.

The site specific in vivo incorporation of p-iodo-phenylalanine andp-bromo-phenylalanine into proteins was performed. Iodine and bromineare heavy atoms, and the incorporation facilitates solving of phaseusing MAD. The site-specific introduction of heavy atoms using unnaturalamino acids also provides selectivity and flexibility in choosingpositions for heavy atoms.

Mutant synthetases with specificities for p-iodo-phenylalanine andp-bromo-phenylalanine, respectively, were generated following themethods and compositions described in Example 1. The protein Z domain(B. Nilsson, et al, Protein Eng. 1:107-113 (1987)) was expressed, inwhich bromine or iodine was selectively introduced in the form ofp-iodo-phenylalanine and p-bromo-phenylalanine using in vivoincorporation of the unnatural amino acids. Protein crystal trays wereset up following standard protocols.

The three dimensional structure of the protein is solved using X-raycrystallography; the phase is determined using the heavy atoms presentin the protein.

Example 7 In Vivo Incorporation of Meta-Tyrosine Analogues

An orthogonal TyrRS was generated for aminoacylation of the mtRNA_(CUA)^(Tyr) (described in Example 1) with meta-tyrosine analogues.

Preparation of mutant TyrRS library plasmids. A library of plasmidsencoding mutant M. jannaschii TryRSs directed at meta-substitutedtyrosine derivatives was constructed, generally following the methodsdescribed in Example 1. Briefly, six residues (Tyr³², Ala⁶⁷, His⁷⁰,Gln¹⁵⁵, Asp¹⁵⁸, Ala¹⁶⁷) in the active site of M. jannaschii TyrRS thatare within 6.9 Å of the meta-position of the aryl ring of bound tyrosinein the crystal structure of Bacillus stearothermophilus TyrRS weremutated to all 20 amino acids at DNA level using the NNK codon scheme asdescribed in Example 1 above. The constructed plasmid library pBK-libcontained around 1×10⁹ independent clones.

Evolution of orthogonal tRNA-synthetase pairs for incorporation ofm-acetyl phenylalanine. After 3 rounds of positive selection and 2rounds of negative selection, five candidate clones (SEQ ID NO: 17-21)emerged whose survival in chloramphenicol was dependent on the additionof the unnatural amino acid. In the absence of m-acetyl phenylalanine,the IC₅₀ of chloramphenicol resistance for cells harboring the one ofthe three mutant TyrRS plasmids is 20 μg/ml. In the presence of m-acetylphenylalanine, the IC₅₀ of resistance to chloramphenicol for the samecells is 100 μg/ml. The large difference between these two numbersreflects the ability of the selected synthetases to specify theincorporation of m-acetyl phenylalanine over the natural amino acids inthe cell. The data for m-methoxy phenylalanine were similar; five cloneswere isolated (SEQ ID NO:22-26).

Protein expression of unnatural amino acid incorporated DHFR. Them-methoxy phenylalanine and m-acetyl phenylalanine synthetases selectedabove were used to incorporate the relevant unnatural amino acids inresponse to an amber codon in DHFR as previously described in Example 1above. As a negative control, cells containing both the orthogonal pairof tRNA-synthetase and amber-mutant vector encoding DHFR were grown inthe absence of unnatural amino acids. The results of protein expressionare shown in FIG. 10. These results clearly demonstrated the specificityof the orthogonal pair of tRNA-synthetase to incorporate unnaturalm-methoxy phenylalanine and m-acetyl phenylalanine. The yields ofexpressed DHFR protein are approximately 0.5 mg/L of culture in bothcases.

Utilizing meta-acetyl phenylalanine as a chemical handle. The m-acetylphenylalanine incorporated DHFR protein was labeled with hydrazidederivatives, both extra-cellularly and intra-cellularly at a milligramscale. The carbonyl group will react rapidly with hydrazide in aqueoussolution to form hydrazone that is stable under physiological conditions(Shao, J.; Tam, J. J. Am. Chem. Soc. 117, 3893-3899 (1995)). Thischemistry has been used by Schultz and coworkers to specifically label aketone containing, purified T4 lysozyme with fluorescein hydrazide(Cornish, V. W.; Hahn, K. M.; Schultz, P. G. J. Am. Chem. Soc. 118,8150-8151 (1996)).

Purified m-acetyl phenylalanine-incorporated DHFR protein was treatedwith fluorescein hydrazide in aqueous buffer. As a control in parallel,a purified m-methoxy phenylalanine-incorporated DHFR protein wassubjected to the same reaction conditions. After the reaction, bothproteins were purified and then excited at 491 nm to obtain fluorescenceemission spectra shown in FIG. 11. Under identical conditions, thepurified m-acetyl phenylalanine-incorporated DHFR was labeled withfluorescein hydrazide while m-methoxy phenylalanine was not labeled.

The fluorescein hydrazide is cell-permeable and does not lyse cells at4° C. Thus, it is possible to label the m-acetylphenylalanine-incorporated DHFR protein intra-cellularly withfluorescein hydrazide. Cells expressing the “ketone handle”-incorporatedDHFR were incubated with fluorescein hydrazide solution. After 36 hoursat 4° C. and extensive washes to remove excess fluorescein hydrazide,the labeled DHFR protein was purified and subjected to fluorescenceemission tests. As a negative control in parallel, m-methoxyphenylalanine-incorporated DHFR was also purified with the sameprocedures. Similar results to the extracellular experiment (FIG. 15)were obtained when intact cells were labeled with fluorescein hydrazideand the DHFRs were subsequently purified.

These experiments demonstrated one example of the utility of a proteinwith at least one unnatural amino acid. Other compounds can be used toin vivo label proteins with at least one unnatural amino acid. Examplesinclude, e.g., biotin hydrazide and other hydrazide derivatives.

Example 8 In Vivo Incorporation of Photoreactive Amino Acids

Introduction: Experiments were performed in which photocrosslinker aminoacids were genetically encoded and site specifically incorporated into aspecific protein in vivo. This protein was then crosslinked at will byexcitation of the photoreactive group-providing temporal control.

This invention is useful for, e.g., exploring protein interactions. Forexample, this invention is useful for defining residues in the proteinprimary sequence that mediate interaction with different cellularcomponents by varying the position of the crosslinker in the protein.Because a covalent bond is formed between the protein and the moleculeit interacts with it is possible to detect weak or transientinteractions.

Two chemical functional groups have gained prominence as crosslinkers,aryl-azides and benophenones since they can be activated at wavelengthsabove 300 nm (below which protein damage via photooxidation may be aproblem). These two crosslinking groups were been incorporated into theunnatural amino acids p-azido-phenylalanine and p-benzoyl-phenylalaninerespectively (FIG. 12).

Generation of O-RS specific for photocrosslinker amino acids. Theorthogonal pair described in Example 1, Methanococcus jannaschiimtRNA_(CUA) ^(Tyr)/TyrRS pair was used as the starting point to generatean O-RS specific for the crosslinker-unnatural amino acidp-azido-phenylalanine (pBpa). The methods for mutagenesis, screening andselection were performed following the experimental outline described inExample 1. Briefly, a MjTyrRS library of mutants was generated in whichfive residues (Tyr 34, Glu 107, Asp 158, Ile 159, Leu 162) wererandomized. These residues were chosen on the basis of the crystalstructure of Bacillus Stearothernophilus TyrRS complexed with tyrosyladenylate (P. Brick, T. N. Bhat & D. M. Blow Journal of MolecularBiology 208, 83 (1989)) in which homologous residues (Tyr34, Asn123,Asp176, Phe177, Leu180) are within 6 Å of the para position of the arylring of bound tyrosine. The mutant TyrRS library was passed through apositive selection based on suppression of an amber stop codon at apermissive site (Asp112) in the chloramphenicol acetyl transferase (CAT)gene. Cells transformed with thesynthetase library, and the CAT mutantwere challenged to grow in the presence of 1 mM pBpa andchloramphenicol. Surviving cells contained synthetases capable ofcharging the orthogonal mtRNA_(CUA) ^(Tyr) with either a natural orunnatural amino acid. These synthetase genes were transferred into cellscontaining mtRNA_(CUA) ^(Tyr) and a variant of the gene encoding thetoxic bamase protein, which contains three amber mutations at permissivesites (Gln2, Asp44, Gly65) (Wang, L., Brock, A., Herberich, B. &Schultz, P. G. Science 292, 498-500 (2001)). Growth of these cells inthe absence of pBpa selected against synthetases capable of utilizingnatural amino acids.

After five rounds of positive and negative selection the survivingsynthetase plasmids were transformed into a reporter strain in which theproduction of full length CAT and T7 RNA polymerase (T7 RNAP) aredependent on suppression of amber stop codons in the CAT and T7 RNAPgene, respectively (Santoro S W, Schultz P G. Proc Natl Acad Sci USAApril 2; 99(7):4185-90 (2002)). Because the T7 RNAP drives expression ofthe green fluorescent protein (GFP) these cells can be fluorometricallyscreened. Ninety-six clones were screened for pBpa dependentchloramphenicol resistance and GFP fluorescence. Six distinctsynthetases conferred Ile chloramphenicol resistance on E. coli withIC₅₀s of 120 mg/L and 5 mg/L in the presence and absence of 1 mM pBparespectively; they also showed pBpa dependent GFP fluorescence. Thelarge difference between the chloramphenicol resistance in the presenceand absence of pBpa shows a substantial in vivo specificity of theselected synthetase/tRNA pairs for insertion of pBpa over all twentynatural amino acids found in the cell in response to an amber codon.

In vivo incorporation of pBpa into myoglobin. To measure the fidelityand efficiency of pBpa incorporation, the codon for Ser4 in sperm whalemyoglobin (containing a C-terminal His6 tag) was converted to an ambercodon. In the presence of both Mj p-BpaRS-1, mtRNA_(CUA) ^(Tyr) andpBpa, full length myoglobin was produced with a purified yield of 2mg/L. No myoglobin protein was detectable by silver stain or Westernblot against the C-terminal His6 tag on myoglobin if any of the threecomponents responsible for specific amber suppression with pBpa (aminoacid, synthetase, or tRNA) were withheld. This data provides furtherevidence that the selected synthetase is very selective for pBpa.

Electrospray-ionization ion trapmass spectrometry of the mutantmyoglobin gave a mass of 18519±0.5 which is identical to the calculatedmass of 18519.0 for the pBpa containing protein. This confirms theincorporation of pBpa at a single site in the protein. No masses wereobserved in the mass spectra corresponding to natural amino acidincorporation providing additional evidence for the high fidelityincorporation of pBp.

Sequence analysis of mutant O-RS. The selected synthetases showinteresting sequence convergence. Tyr32 of M. jannaschii TyrRS isconverted to alanine or glycine in five of the six mutant synthetaseclones. Asp158 of the M. jannaschii TyrRS is converted to threonine infive of the six selected mutants, while Ile159 is converted to serine infour of the six mutants. Serine or proline substitutions dominate atposition 107 of M. jannaschii TyrRS; Leu162 is conserved in four of thesix mutants. A consensus set of mutations (32:Gly, Ala/107:Ser,Pro/158:Thr/159:Ser/162:Leu) emerges from this analysis.

In vivo incorporation of pBpa into GST. To demonstrate the utility ofthis methodology for mapping protein-protein interactions, a cosslinkingexperiment was carried out with glutathione-S-transferase. This proteinis a dimer of two identical subunits which have previously beencrosslinked non-specifically using gluteraldehyde. The crystal structureof the dimeric Schistosoma Japonica glutathione-S-transferase (SjGST)(McTigue, M. A., Williams, D. R. & Tainer, J. A. Journal of MolecularBiology 246, 21-27 (1995)) was used to identify two sites to substitutewith pBp: residue Phe52, which is buried in the dimer interface of thecrystal structure, and residue Tyr198 which is solvent exposed. Thecodons corresponding to Phe52 or Tyr198 in the gene for a 27 kDa proteinSj GST, were replaced with amber codons. The orthogonal synthetase tRNApair was then used to site specifically incorporate pBpa into SjGST inE. coli at these sites. Upon irradiation with long wavelengthultraviolet radiation, purified SjGST was converted to a covalentlylinked homodimer as judged by denaturing SDS PAGE. Approximately 70% ofthe SjGST present was crosslinked in 5 minutes. In contrast, controlexperiments using either wild type SjGST or SjGST containing pBpa atresidue 198, which lies outside the dimer interface, shows no detectablecrosslinking in response to UV irradiation.

These results demonstrate that site-specific pBpa substitution can beused to define amino acids involved in a protein-protein interaction.

Characterization of Mutant Synthetases Individual synthetase clones inDH10B/pREP(2)/YC-JYCUA were used to inoculate 0.5 mL of LB supplementedwith kanamycin and tetracycline to 30, 20 mg/L. After 20 hours growth(37° C., 300 rpm) cells were diluted 10₄ fold in d_(H)20 and replicaspotted on two sets of GMML plates. One set of plates were supplementedwith kanamycin and tetracycline at 30 and 20 micrograms/L, respectively,and chloramphenicol at concentrations ranging from 0 micrograms/L to 110micrograms/L. The second set of plates were identical to the first,except that they were supplemented with 1 mM pBpa. After 48 h theI_(C50) of chloramphenicol resistance in the presence and absence ofpBpa was calculated from the concentration of chloramphenicol at whichhalf the number of colonies on the plates with no chloramphenical werevisible. GFP expression in the presence and absence of pBpa was imagedusing a Storm phosphoimager (Molecular dynamics). Mutant synthetasegenes exhibiting the strongest amino acid dependence in both GFP signaland chloramphenicol resistance were isolated and sequenced by standardmethods.

Protein Expression Plasmid PYC/SjGSTmut, which contains the mutant SjGSTgene on an arabinose promoter and rrnB terminator, and mtRNAT_(CUA)^(Tyr) on a lpp promoter and rmC terminator, and a tetracyclineresistance marker was co-transformed with a pBK vector expressingp-BpaRS into DH10B E. coli. Cells were amplified in 10 mL of 2×-YTcontaining kanamycin at 30 micrograms/L and tetracycline at 25micrograms/L before being washed in PBS and used to inoculate 1 L ofliquid GMML with the appropriate antibiotics and pBpa to 1 mM. Proteinexpression was induced at an OD600 of 0.6 by the addition of arabinoseto 0.2% followed by 5 hours growth. Cells were harvested bycentrifugation and protein was purified by virtue of a C-terminalhexa-histidine tag using Ni-NTA affinity chromatography.

Sperm whale myoglobin was expressed and purified from cells containingpBAD/JYAMB-4TAG in an analogous manner to SjGST, except that inductionwas constitutive with 0.002% arabinose. Samples for mass spectrometrywere desalted on a NAP-10 column (Pharmacia) and purified by HPLC. Toverify the incorporation of pBpa, the protein mass was ascertained byelectrospray-ionization ion trap mass spectrometry.

Mutant Sj GST Cloning Mutant SjGST genes were assembled by overlappingPCR, using pGEX-3 (Pharmacia) as a template. All PCR reactions werecarried out using the Expand PCR kit (Roche) according to themanufacturers instructions. The resulting genes were digested with Nco Iand Kpn I restriction enzymes and cloned into predigested,dephosphorylated pBADJYC vector between the same restriction sites andin frame with a C-terminal hexa-histidine tag. All final constructs wereconfirmed by DNA sequencing.

Photo-activated crosslinking. Crosslinking reactions were performed in a96 well microtitre plate (Nuncsorb) using 100 μL of 10 ng/μL SjGST (in50 mM NaH2Po4, 300 mM NaCl, 250 mM imidazole) at 4° C. Samples wereirradiated at 365 nm using a handheld UV lamp (1 SV, 60 Hz, 0.2 A;Spectronics, NY, USA), for 1 min or 5 min. Samples were removed from thewells and diluted with SDS loading buffer before resolution of productsby SDS-PAGE on a 10-20% gradient gel. SjGST was transferred to PVDF(Biorad) and probed by western blot using goat anti-GST (Pharmacia) anda secondary mouse anti goat HRP conjugate (Sigma). Signal was developedusing Super signal West (Pierce) and visualized by exposure on hyperfilm(Amersham).

Example 9 Synthesis of Meta-Substituted Phenylalanines

In one aspect, the present invention provides meta substitutedphenylalanines as shown in Formula IV:

and in Formula V.:

Formula IV illustrates the structure of 3-acetyl-phenylalanine andFormula V represents 3-methoxy-phenylalanine.

Meta-substituted phenylalanines are synthesized in a procedure asoutlined in FIG. 14. Typically, NBS (N-bromosuccinimide) is added to ameta-substituted methylbenzene compound to give a meta-substitutedbenzyl bromide, which is then reacted with a malonate compound to givethe meta substituted phenylalanine. Typical substituents used for themeta position include, but are not limited to, ketones, methoxy groups,alkyls, acetyls, and the like. A specific example is provided below.

NBS (N-bromosuccinimide) was recrystallized from boiling water prior tousage. NBS (1.85 g, 10.5 mmol) was added to a solution of 3-methylacetophone (1.34 g, 10 mmol). AIBN (2′,2′-azobisiosbutyronitrile) (0.043g, 0.25 mmol) was added to the mixture. The reaction mixture wasrefluxed for 4 hours. The completion of reaction was checked by TLC(8:1/hexanes:EtOAc). After aqueous workup, the organic solvent wasremoved and hexanes was added to give solid. The solid was filtered andwashed with hexanes and EtOAc. Then the mixture was recystallized withhexanes. The supernatant was collected and solvent was removed to givecompound (1-(3-bromomethyl-phenyl)-ethanone).

Dry ethanol (50 ml) was added dropwise to pentane-washed sodium pieces(2.3 g, 0.1 mol) under argon atmosphere. After the completion ofaddition, stirring was required to dissolve the last pieces of sodium. Asolution of diethyl acetylamido-malonate ester (21.7 g, 0.1 mol) wasadded over 30 minutes. 1-(3-bromoethyl-phenyl)ethanone (21.1 g, 0.1 mol)in dry ethanol was added dropwise over 90 minutes. After the mixture wasrefluxed overnight, ether and water was added, and the organic layer wasseparated. After aqueous workup, the organic layers were combined,washed with brine, dried over MgSO4 and filtered. The solvents wereremoved in vacuo. Hexanes-dichloromethane, 4:1, was added to theresidue, and the insoluble material was filtered out and washedexhaustively with 10:1 dicholomethane-benzene to give diethyl2-acetamido-2[3-acetyl-phenyl]-methyl]malonate. This compound wasstirred with 8 M HCl in dioxane overnight. Then the mixture was taken todryness, water was added, and it was taken to dryness again to givefinal compound m-acetylphenylalanine hydrochloride. HPLC was used topurify the desired compound as white solid. The total yield was 64%.¹HNMR (D2O): d 7.85-7.28 (m, 4H), 4.23 (dd, 1H), 3.2 (m, 2H), 2.7 (s,3H). Calculated molecular weight: 243.69, obtained molecular weight:243.07. A similar synthesis is used to produce a 3-methoxyphenylalanine. The R group on the meta position of the benzyl bromide inthat case is —OCH₃. See, e.g., Matsoukas et al., J. Med. Chem., 1995,38, 4660-4669.

Example 10 Synthesis of 4-allyl-L-tyrosine

In another aspect, the present invention provides 4-allyl-L-tyrosine,whose structure is shown in Formula II:

The compound of Formula II, 4-ally-L-tyrosine, is synthesized accordingto the scheme set forth in FIG. 13. A protected tyrosine, e.g., an Nbocor Fmoc protected tyrosine, is reacted with allyl bromide, resulting ina protected allyl tyrosine, which is then typically deprotected to yield4-allyl-L-tyrosine. For example, N-(tert-Butoxycarbonyl)-L-tyrosine(2.95 g, 10 mmole) was dissolved in 80 ml of DMF. The solution waschilled to 5° C. and NaH (0.63 g, 26 mmole) was added. The reactionmixture was allowed to warm up to 10° C. and stirred for additional 2hours. After that, allyl bromide (1.33 g, 11 mmole) was added to themixture and reaction was warmed to room temperature. The reactionmixture was stirred for 4 hours. Water was added to work up thereaction. The aqueous layer was extracted with ethyl acetate and CH2Cl2.The organic layer was dried over anhydrous MgSO4. The organic solventwas removed to give white solid. This compound was then refluxed in 4MHCl in 1,4-dioxane for 4 hours. All the solvent was evaporated to givethe desired product as white solid (1.9 g, 86%). 1HNMR (CD3OD): d ppm3.1 (m, 2H), 4.1 (t, 1H), 4.5 (d, 2H), 5.3 (q, 1H), 5.9 (m, 1H), 6.9 (d,2H), 7.1 (d, 2H). Calculated molecular weight: 221, obtained molecularweight: 222.

Example 11 Cellular Uptake Screen of Unnatural Amino Acids

A variety of unnatural amino acids and α-hydroxy acids of interest,obtained commercially or by short syntheses from available startingmaterials (I. Shin, B. Herberich, A. Varvak, T. Magliery, P. Schultz,unpublished results, were screened for cell toxicity). For example, FIG.29 provides a library of unnatural amino acids useful for the followingscreen. Each amino acid wasscreened at 1 mM in glycerol minimal mediafor toxicity to cells, e.g., to DH10B harboring pBLAM-YQRS andpACYsupA38. Toxicities are sorted into five groups: (1) no toxicity, inwhich no significant change in doubling times occurs; (2) low toxicity,in which doubling times increase by less than about 10% (seen with thefollowing compounds in FIG. 29: S63, S69, S74, S75, S81, S95); (3)moderate toxicity, in which doubling times increase by about 10% toabout 50% (seen in the following compounds shown in FIG. 29: B, M, P,S12, S14, S22, S41, S45, S49, S52, S62, S64, S65, S71, S91, S93, B10);(4) high toxicity, in which doubling times increase by about 50% toabout 100% (seen in the following compounds from FIG. 29: C, Q, V, BB,S2, S5, S50, S60, S78, S83, S89, S90); and (5) extreme toxicity, inwhich doubling times increase by more than about 100% (observed for thefollowing compounds from Figure 29: W, S15, S26, S27, S30, S31, S39,S47, S88, S94). See, e.g., Liu, D. R. & Schultz, P. G. Progress towardthe evolution of an organism with an expanded genetic code. Proceedingsof the National Academy of Sciences of the United States of America 96,4780-4785 (1999).

The toxicity of amino acids scoring as highly or extremely toxic istypically measured as a function of their concentration to obtain IC50values. In general, amino acids which are very close analogs of naturalamino acids (e.g., Q, W, S5, S26, S27, S50, S90, S94) or which displayreactive functionality (e.g., S15, S39, S47) demonstrated the highesttoxicities.

To identify possible uptake pathways for toxic amino acids, toxicityassays were repeated at IC50 levels (typically 3 μM to 500 μM) in mediasupplemented with an excess (2 mM) of a structurally similar naturalamino acid. For toxic amino acids, the presence of excess natural aminoacid rescued the ability of the cells to grow in the presence of thetoxin, presumably because the natural amino acid effectively outcompetedthe toxin for either cellular uptake or for binding to essentialenzymes. In these cases, the toxic amino acid can be assigned a possibleuptake pathway and labeled a “lethal allele” whose complementation isrequired for cell survival. Lethal alleles identified in this manner (16of the toxic unnatural amino acids) span ten possible amino acid uptakegroups: alanine, glutamic acid, lysine, leucine, methionine, proline,glutamine, arginine, threonine, and tyrosine.

These lethal alleles are extremely useful for assaying the ability ofcells to uptake nontoxic unnatural amino acids. Each nontoxic unnaturalamino acid was added at 2 mM to media containing IC50 levels of eachlethal allele. Complementation of the toxic allele, evidenced by therestoration of cell growth, shows that the nontoxic amino acid is takenup by the cell, possibly by the same uptake pathway as that assigned tothe lethal allele. A lack of complementation is inconclusive.

Using this method, the ability of 22 glutamine and glutamic acid analogsto be taken up by DH10B was evaluated. Amino acids S27 and S47 were usedas toxic glutamine alleles at 100 μM and 30 μM, respectively, while S50was employed as a toxic glutamic acid allele at 150 μM. Results from S27and S47 complementation were in complete agreement and identified aminoacids B, Z, S6, S60, S61, and S62 (in addition to S27 and S47) as beinguptaken by cells possibly via the glutamine uptake pathway. Similarly,complementation of S50 identified B, C, K, X, S60, S65, and S84 as beinguptaken into DH10B, possibly via the glutamic acid transport system.

These findings indicate that the E. coli glutamine and glutamic acidtransport pathways may tolerate significant perturbations in amino acidstructure, including side chain elongation (X and Z), ketone ormethylene placement at the y-position (B, C, S65), carboxamidereplacement with a sulfoxide (S61), a known substrate for a bacterialglutamine transporter or hydrazide (S47), also a known glutaminetransporter substrate as well as a variety of hybridization changes atthe side chain terminus (S60, S62, K, S84). See, e.g., Jucovic, M. &Hartley, R. W. Protein-protein interaction: a genetic selection forcompensating mutations at the barnase-barstar interface. Proceedings ofthe National Academy of Sciences of the United States of America 93,2343-2347 (1996) and Weiner, J. H., Furlong, C. E. & Heppel, L. A. Abinding protein for L-glutamine and its relation to active transport inE. coli. Archives of Biochemistry and Biophysics 142, 715-7 (1971).

Example 12 Biosynthesis of β-Aminophenylalanine

To produce the unnatural amino acid p-aminophenylalanine (pAF) in vivo,genes relied on in the pathways leading to chloramphenicol andpristinamycin are optionally used. For example, in StreptomycesVenezuelae and Streptomyces pristinaespiralis, these genes produce pAFas a metabolic intermediate. See, e.g., Yanai, K. and e. al.,Streptomyces venezuelae genes papA, papB, papC, in PCT Int. Appl. 2001,Meiji Seika Kaisha Ltd.: Japan. p. 1-83; and Blanc, V., et al.,Identification and analysis of genes from Streptomyces pristinaespiralisencoding enzymes involved in the biosynthesis of the4-dimethylamino-L-phenylalanine precursor of pristinamycin I. MolecularMicrobiology, 1997. 23(2): p. 191-202.

A biosynthetic pathway for pAF is shown in FIG. 15, Panel B. pAF isoptionally synthesized in E. coli from chorismate (compound 2 in FIG.15, Panel B), which is a biosynthetic intermediate in the synthesis ofaromatic amino acids. To synthesize pAF from chorismate, a celltypically uses a chorismate synthase, a chorismate mutase, adehydrogenase, e.g., a prephenate dehydrogense, and an aminotransferase. For example, using the S. Venezuelae enzymes PapA, PapB,and PapC together with an E. coli aminotransferase, e.g., as shown inFIG. 15, Panel B, PapA, chorismate is used to produce pAF.

For example, 4-amino-4-deoxychorismate synthase converts chorismate to4-amino-4-deoxychorismic acid (compound 3 in FIG. 15, Panel B), e.g.,using ammonia (from glutamine) in a simple addition-eliminationreaction. PapB and PapC, which are analogous to chorismate mutase andprephenate dehydrogenase, respectively, are used to convert4-amino-4-deoxychorismic acid to 4-amino-4-deoxyprephenic acid (compound4 in FIG. 15, Panel B) and then to p-aminophenyl-pyruvic acid (compound5 in FIG. 15, panel B). A non-specific tyrosine aminotransferase, e.g.,from E. coli is used to convert p-aminophenyl-pyruvic acid to pAF. See,e.g., Escherichia coli and Salmonella, 2nd ed, ed. F. C. Neidhardt.Vol. 1. 1996, Washington, D.C.: ASM Press. For example, tyrB, aspS, orilvE is optionally used to produce pAF from p-aminophenyl-pyruvic acid.

FIG. 13 illustrates a plasmid for use in the biosynthesis of pAF. Theplasmid depicted comprises S. Venezuele genes papA, papB, and papCcloned into a pSC101 derived pLASC plasmid, e.g., under control of thelac or lpp promotor. The plasmid is used to transform a cell, e.g., abacterial cell, such that cell produces the enzymes encoded by thegenes. When expressed, the enzymes catalyze one or more reactionsdesigned to produce a desired unnatural amino acid, e.g., pAF. Forexample, proteins PapA, PapB and PapC convert chorismate top-aminophenyl-pyruvic acid, while an E. coli aromatic aminotransferasecompletes the biosynthesis to afford pAF.

Typically, the synthesis of pAF from chorismate, in the presentinvention does not affect the concentration of other amino acidsproduced in the cell, e.g., other aromatic amino acids typicallyproduced from chorismate. Typically, p-aminophenylalanine is produced ina concentration sufficient for efficient protein biosynthesis, e.g., anatural cellular amount, but not to such a degree as to affect theconcentration of the other aromatic amino acids or exhaust cellularresources. Typical concentrations of pAF produced in vivo in this mannerare about 10 mM to about 0.05 mM. In S. Venezuelae evidence suggeststhat the regulation of the shikimate pathway is modified to account forchorismate consumption in making a fourth aromatic amino acid. See,e.g., He, J., et al., Microbiology, 2001 147: p. 2817-2829. Once abacterium is transformed with the plasmid comprising the genes used toproduce enzymes used in the above pathway, and pAF as a twenty-firstamino acid is generated, in vivo selections are optionally used tofurther optimize the production of pAF for both ribosomal proteinsynthesis and cell growth.

Since a pAF tRNA-synthetase pair allows the suppression of a TAG codonin a nonessential position of a protein, biosynthetic pathwayeffectiveness is optionally monitored and optimized by the production ofthat protein. Only cells that produce a concentration of pAF sufficientfor protein biosynthesis are able to suppress the TAG codon. At the sametime, one can select for optimal pAF production based on E. coli growthrates if the TAG-protein is an essential protein to cell growth. Placingthe biosynthetic genes on a plasmid allows the level of pAF produced tobe modified, e.g., by changing plasmid copy number and promotorstrength. To determine if the addition of a pAF biosynthetic pathwayaffects the production of other aromatic amino acids in E. coli, and toquantitte pAF production, the cellular concentrations of the aromaticamino acids is optionally monitored, e.g., by extraction and LCMSanalysis. See, e.g., Moss, R. E., Methods in Enzymology, 1995. 262: p.497-499 and Mimura, H., S, Nagata, and T. Matsumoto, Biosci. Biotech.Biochem., 1994. 58(10): p. 1873-1874

Example 13 Biosynthesis of Dopa

To biosynthetically produce dopa in vivo, one or more genes, e.g.,hpaBC, for a nonspecific aromatic hydroxylase, e.g., from E. coli arecloned into a low copy number vector, e.g., a pSC101 derivative, whichis typically placed under control of an lpp promotor. This constructproduces dopa (2) from tyrosine (1), in vivo, as shown in FIG. 20 whilenot being toxic to the growing cells. Similar work was done with thisgene to overproduce dopa for purification purposes. See, e.g.,Jang-Young Lee, Luying Xun, Biotechnology Letters, 1998, 20, 479-482.However, as described above, overproduction is not typically desired. Inthis application, a low copy plasmid is used to produce dopa in anatural cellular amount.

Example 14 Biosynthesis of O-methyl-L-tyrosine

O-methyl-L-tyrosine is optionally produced biosynthetically by plantO-methyltransferases are enzymes involved in secondary metabolism, whichconverts a hydroxyl group into a methoxyl group. Two such enzymes wereselected: (iso)eugenol O-methyltransferase (IEMT) and caffeic acidO-methyltransferase (COMT). Both of them are from Clarkia breweri. IEMTmethylates eugenol/isoeugenol, and COMT methylates caffeic acid. Thesubstrates of these two enzymes are similar to tyrosine. However, bothenzymes have high substrate specificity and methylationregiospecificity. Therefore, a combinatorial approach to evolve thesetwo enzymes was adopted so that they would take tyrosine as theirsubstrate and convert tyrosine into O-methyl-L-tyrosine. Active siteresidues were selected for mutation, and large mutant libraries werecreated. After several rounds of selection, at least about three hitshave been identified.

In other embodiments, the enzymes used to produce O-methyl-L-tyrosinecan also be artificially evolved, e.g., to produce a meta substitutedmethoxy phenylalanine as provided in Formula III.

Example 15 Biosynthesis of Glycosylated Amino Acids

The present invention also provides biosynthetic methods for theproduction of glycosylated amino acids. Forming glycosylated amino acidsin vivo is optionally performed in a number of ways. For example,transforming a cell with a plasmid comprising a gene for aN-acetyl-galactosaminidase, a transglycosylase, or a hydralase, e.g.,serine-glycosyl hydrolase, e.g., acting in the reverse direction,provides a cell that produces a glycosylated amino acid. When combinedwith a translation system as provided below, the biosynthetic pathwayresults in a cell that produces and incorporates a glycosylated aminoacid into one or more proteins within the cell. For example, see, e.g.,FIG. 28, illustrating the formation of a glycosylated amino acid,wherein R is optionally an alcohol, an amine, or an N-acetyl amine. Anexample structure is shown by Formula IV:

Example 16 Identification of Advantages Due to Incorporation ofUnnatural Amino Acids

Given the capability presented herein of developing a completelyautonomous bacterium that can biosynthesize a unnatural amino acid frombasic carbon sources and incorporate this amino acid into proteins,e.g., in response to a nonsense codon in DNA, with high translationalefficiency and fidelity, the question remains whether such additionsactually provide an advantage to the bacterium over an organism thatincorporates only the twenty natural amino acids. The present inventionprovides a method of determining if an expanded genetic code providesany such advantage as well as identifying the type of advantage and theunnatural amino acid to which it is due.

Since the 19th century bacteriologists have been interested in theextraordinary changes of bacterial cultures grown under variousconditions. See, e.g., Summers, W. C., J. Hist. Biol., 1991, 24: P.171-190. However, all forms of evolution have been studied with twentyamino acid organisms. The present invention addresses the feasibility ofexpanding the genetic code of E. coli with unnatural amino acids andprovides methods of testing whether the ability to incorporateadditional amino acids provides E. coli with an evolutionary advantage.

To determine whether the addition of novel amino acids to the geneticcode can provide an evolutionary advantage to E. coli. the evolution ofa twenty-one amino acid bacteria is optionally compared to that of atwenty amino acid bacteria. The approach combines new sets oftranslational machinery for incorporation of unnatural amino acids intoproteins with a mutagenized E. coli genomic library placed underselective pressures. The genetic, selection approach described above andelsewhere by Schultz and coworkers has, thus far, produced at leastabout eleven new aminoacyl synthetases that can incorporate novel aminoacid into proteins efficiently and with high fidelity in response to theTAG codon.

Mutagenizing a plasmid library of the E. coli genome scrambles codonsand randomly adds TAG nonsense codons throughout the genome. The new TAGcodons can be suppressed by the incorporation of new amino acids intothe expressed protein. These bacterial systems are placed underselective pressures to select for enhanced E. coli growth. See, e.g.,FIG. 21. The selected genomic fragments from the library that confer anadvantage are optionally isolated and screened for enhanced growthability when incorporating the other unnatural amino acids and tyrosinein response to TAG codons.

A pSC101 low copy vector (approximately 5 copies/cell) is optionallyused to construct a large insert (7-14 kb) E. coli genomic library bystandard methods known to those of skill in the art. For example, a 600member pSC101 based E. coli genomic library provides complete coverageof the E. coli genome and is also compatible with the aminoacylsynthetase, and tRNA plasmids described above. Many mutagens have beenstudied for there ability to incorporate TAG codons into genes. See,e.g., Miller, J. H., A short course in bacterial genetics. 1992,Plainview: Cold Spring Harbor Laboritory Press. Multiple mutagenesismethods are optionally used since each mutagen is not completely randomin its formation of TAG codons. By mutagenizing the same 600 member E.coli genomic library with four different mutagens TAG codons aretypically placed in as many sites as possible. Four optional methodsinclude, but are not limited to, UV irradiation, a mutator strain (XL1red), 4-nitro-quinoline-1-oxide (NQO), and ethylmethane sulfonate (EMS)to mutate the genomic library, and combine them to make one largemutated genomic libraries of >1010 members. These mutation methods allrely primarily on forming point mutation but complement each other inthe mechanism of mutagenesis resulting over all in a more evendistribution of TAG codons. UV irradiation and the mutator straingenerate all base substitutions while NQO and EMS principally cause G:Cto A:T transitions. Most of the point mutations generated form codonsthat code for one of the twenty natural amino acids. Since only about12.5% of the single point mutations can form a TAG codon large highlymutagenized genomic libraries are needed. This method typicallygenerates a least about 106 mutated copies of each gene with many newrandomly placed codons. The genomic library is then typically checkedfor TAG codon incorporation by sequencing a subset of library membersbefore and after mutagenesis.

To determine which genes might be improved by incorporation of one ofthe new amino acids any of a variety of selective pressures areoptionally used for screening that target a range of cellular biology:catalytic functions, protein interactions, carbon sources, multipleresponse genes, and broad metabolic functions. For example, selectionpressure based on quinolones is used to target topoisomerase and DNAgyrase. 5-fluorouracil is used to target DNA synthesis; omeprazole isused to target proton pump inhibitors; the use of fatty acids as a solecarbon source and acidic media are used to target a variety of genesrelated to utilization of carbon and response; and a reductive media isused to target the thiol-redox pathway and disulfide containingproteins. See, e.g., Bronson, J. J. and J. F. Barrett, Curr. Med.Chemistry, 2001 8: p. 1775-1793; Bearden, D. T. and L. H. Danziger,Pharmacotherapy, 2001 21(10): p. 224S-232S; Matthews, D. A., et al., J.Mol. Biol., 1990. 2144(4): p. 937-948; Knox, M. R. and J. E. Harris,Arch. Microbiol., 1988. 149(6): p. 557-60; McGowan, C. C., T. L. Cover,and M. J. Blaser, Gasteroenterology, 1994. 107(5): p. 1573-8; Clark, D.P. and J. E. Cronan, Two carbon compounds and fatty acids as carbonsources. Escheria coli and Salmonella cellular and molecular biology,ed. F. C. Neidhardt. Vol. 1. 1996, Washington D.C.: ASM press;Slonczewski, J. L. and J. W. Foster, pH-regulated genes and survival atextreme pH. Escheria coli and Salmonella cellular and molecular biology,ed. F. C. Neidhardt. Vol. 1. 1996, Washington D.C.: ASM press; and Ritz,D. and J. Beckwith, Annu. Rev. Microbiol., 2001. 55: p. 21-48.

The screening of the mutated genomic library produces a set of mutatedgenomic fragments that confer a growth advantage under a certainselection pressure. These fragments are compared to determine if theyare the same found in screens with no unnatural amino acid present byrestriction mapping and sequencing. Fragments that produce a growthenhancement from an unnatural amino acid selection this fragment areoptionally re-screened by comparing growth rate with each unnaturalamino acid and tyrosine suppressing the TAG codon. See, e.g., FIG. 21.This re-screening of selected genomic fragments insures that theunnatural amino acid is the factor in conferring a growth advantage. Forfragments that show a selective growth advantage with an unnatural aminoacid being inserted into TAG codons, the gene(s) that confers anadvantage is optionally isolated and identified, e.g., by digestion andsubcloning. The protein can be studied to identify how the unnaturalamino acid is enhancing cellular function. The enhanced protein isoptionally purified and compared to a natural protein with both in vitroand in vivo studies. Standard enzyme techniques are optionally used tostudy protein stability, kinetics, and its interaction with otherbiosynthetic pathway components.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1-47. (canceled)
 48. A method for producing in a translation system aprotein on the surface of a bacterial cell wherein said proteincomprises at least one unnatural amino acid, the method comprising:providing the translation system with at least one nucleic acid encodingsaid protein comprising at least one selector codon, wherein the nucleicacid encodes at least one Omp-A protein or portion thereof; providingthe translation system with an orthogonal tRNA (O-tRNA), wherein theO-tRNA functions in the translation system and wherein the O-tRNArecognizes the at least one selector codon; providing the translationsystem with an orthogonal aminoacyl tRNA synthetase (O-RS), wherein theO-RS preferentially aminoacylates the O-tRNA with the at least oneunnatural amino acid in the translation system; and providing thetranslation system with the at least one unnatural amino, therebyproducing in the translation system the at least one protein comprisingthe at least one unnatural amino acid. 49-50. (canceled)
 51. The methodof claim 50, wherein the translation system comprises an Escherichiacoli cell.
 52. The method of claim 49, wherein the translation systemcomprises an archeaebacterial cell.
 53. The method of claim 49, whereinthe translation system comprises a eukaryotic cell. 54-59. (canceled)60. The protein of claim 55, wherein the protein comprises at least twounnatural amino acids.
 61. The protein of claim 55, wherein the proteincomprises at least three unnatural amino acids.
 62. The protein of claim55, wherein the protein comprises at least four unnatural amino acids.63. The protein of claim 55, wherein the protein comprises at least fiveor more unnatural amino acids.
 64. The method of claim 48, wherein thetranslation system comprises an in vitro translation system.
 65. Themethod of claim 64, wherein the translation system comprises a cellextract. 66-67. (canceled)
 68. The method of claim 48 wherein the atleast one unnatural amino acid is a O-methyl-L-tyrosine.
 69. The methodof claim 48 wherein the at least one unnatural amino acid is anL-3-(2-naphthyl)alanine.
 70. The method of claim 48 wherein the at leastone unnatural amino acid is an amino-, isopropyl-, or O-allyl-containingphenylalanine analogue.
 71. The method of claim 48, wherein the O-tRNAcomprises a nucleic acid comprising a polynucleotide sequence selectedfrom the group consisting of: SEQ ID NO: 1-3 and a complementarypolynucleotide sequence thereof.
 72. The method of claim 48 wherein theat least one selector codon is nonsense codon, a rare codon, or a fourbase codon.
 73. The method of claim 72 wherein the nonsense codon is anamber codon.
 74. The method of claim 48, wherein the O-RS preferentiallyaminoacylates the O-tRNA with an O-methyl-L-tyrosine.
 75. The method ofclaim 48, wherein the O-RS preferentially aminoacylates the O-tRNA withan amino-, isopropyl-, or O-allyl-containing phenylalanine analogue. 76.The method of claim 48, wherein the O-RS comprises a polypeptideselected from the group consisting of: a polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 4-34;and, a polypeptide encoded by a nucleic acid comprising a polynucleotidesequence selected from the group consisting of: SEQ ID NO:35-66 and acomplementary polynucleotide sequence thereof.
 77. The method of claim48 wherein the unnatural amino acid is provided exogenously.
 78. Themethod of claim 48 wherein the translation system is a cell and theunnatural amino acid is biosynthesized by the cell. 79-140. (canceled)